Inhibition of cardiac fibrosis in myocardial infarction

ABSTRACT

The described invention provides a method for treating myocardial infarction (MI) in a subject comprising administering to the subject a therapeutic amount of a pharmaceutical composition comprising a polypeptide of amino sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent thereof made from a fusion between a first polypeptide that is a cell permeable protein (CPP) selected from the group consisting of a polypeptide of amino acid sequence YARAAARQARA (SEQ ID NO: 2), WLRRIKAWLRRIKA (SEQ ID NO: 21), WLRRIKA (SEQ ID NO: 22), YGRKKRRQRRR (SEQ ID NO: 23), FAKLAARLYR (SEQ ID NO: 25), and KAFAKLAARLYR (SEQ ID NO: 26), and a second polypeptide that is a therapeutic domain (TD), and a pharmaceutically acceptable carrier. The described invention also provides a kit comprising a composition comprising at least one MK2 inhibitor peptide; a means for administering the composition; and a packaging material.

This application is a continuation of U.S. patent application Ser. No.14/255,643, filed on Apr. 17, 2014 and issued as U.S. Pat. No.10,336,788, the disclosure of which is hereby incorporated by referencein its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MOER-010-01US_SeqList.txt, date recorded: Jul. 2, 2019, file size 5,832bytes).

FIELD OF THE INVENTION

The described invention relates to the inhibition of cardiac fibrosis inmyocardial infarction by direct actions on cardiomyocytes andfibroblasts by inhibiting Mitogen Activated Protein Kinase ActivatedProtein Kinase II (MK2) activity.

BACKGROUND OF THE INVENTION Ischemic Heart Disease/Myocardial Infarction

Ischemic heart disease is the most common cause of death in the world.In the United States alone an estimated 785,000 people will have amyocardial infarction (MI) each year; approximately 1 per minute [RogerV L, Go A S, Lloyd-Jones D M, Benjamin E J, Berry J D, Borden W B, etal. Executive summary: heart disease and stroke statistics—2012 update:a report from the American Heart Association. Circulation. 2012;125:188-97]. The adverse remodeling that occurs after myocardialinfarction contributes to the impaired cardiac function and heartfailure associated with increased morbidity and mortality. Advances madein interventional, largely early reperfusion therapies, have improvedpatient survival while increasing the morbidity and mortality of theresulting heart failure [Pfeffer M A, Braunwald E. Ventricularremodeling after myocardial infarction. Experimental observations andclinical implications. Circulation. 1990; 81:1161-72; Opie L H,Commerford P J, Gersh B J, Pfeffer M A. Controversies in ventricularremodeling. Lancet. 2006; 367:356-67; Dorn G W, 2nd. Novelpharmacotherapies to abrogate post-infarction ventricular remodeling.Nat Rev Cardiol. 2009; 6:283-91]. The size of the infarcted area, theinfarcted wound healing, and chronic left ventricular (LV) remodelingdetermine the extent of heart failure that results [Pfeffer M A,Braunwald E. Ventricular remodeling after myocardial infarction.Experimental observations and clinical implications. Circulation. 1990;81:1161-72; Opie L H, Commerford P J, Gersh B J, Pfeffer M A.Controversies in ventricular remodelling. Lancet. 2006; 367:356-67; DornG W, 2nd. Novel pharmacotherapies to abrogate post-infarctionventricular remodeling. Nat Rev Cardiol. 2009; 6:283-91].

Ischemia

The myocardium depends almost entirely on aerobic metabolism, sinceoxygen stores in the heart are meager. Myocardial oxygen supply risesand falls in response to the oxygen (energy) demands of the myocardium.The term “autoregulation” refers to the ability to maintain myocardialperfusion at constant levels in the face of changing driving forces.Auto-regulation maintains coronary perfusion at relatively constantlevels over a wide range of mean aortic pressure. When aortic pressureexceeds its upper or lower limits, coronary blood flow precipitouslydeclines or increases proportionately.

The heart needs to be supplied with a sufficient quantity of oxygen toprevent under-perfusion. When reduced perfusion pressure distal tostenoses is not compensated by auto-regulatory dilation of theresistance vessels, ischemia, meaning a lack of blood supply and oxygen,occurs. Because the zone least supplied generally is the farthest out,ischemia generally appears in areas farthest away from the blood supply.

After total or near-total occlusion of a coronary artery, myocardialperfusion occurs by way of collaterals, meaning vascular channels thatinterconnect epicardial arteries. Collateral channels may form acutelyor may preexist in an under-developed state before the appearance ofcoronary artery disease. Preexisting collaterals are thin-walledstructures ranging in diameter from 20 μm to 200 μm, with a variabledensity among different species. Preexisting collaterals normally areclosed and nonfunctional, because no pressure gradient exists to driveflow between the arteries they connect. After coronary occlusion, thedistal pressure drops precipitously and preexisting collaterals openvirtually instantly.

The term “myocardial ischemia” refers to a decrease in blood supply andoxygen to the cells of the myocardium. The development of myocardialischemia has been attributed to two mechanisms: (1) increased myocardialoxygen demand, and (2) decreased myocardial perfusion and oxygendelivery [Willerson, J. T. et al., J. Am. Coll. Cardiol. 8(1): 245-50(1986)]. Myocardial ischemia generally appears first and is moreextensive in the subendocardial region, since these deeper myocardiallayers are farthest from the blood supply, with greater need for oxygen.

Transient Ischemia

The term “transient ischemia” as used herein refers to a reversible(meaning that the myocytes survive the insult) narrowing of a coronaryartery at rest or with exercise where there is no thrombus or plaquerupture but where blood supply cannot be met. Every time the heart'soxygen demand increases, an imbalance between oxygen demand and supplyis created. Transient ischemia produces a cascade of events beginningwith metabolic and biochemical alterations leading to impairedventricular relaxation and diastolic dysfunction, impaired systolicfunction, and electrocardiographic abnormalities with ST segmentalterations, followed by increased end-diastolic pressure with leftventricular dyssynchrony, hypokineses, akinesis, and dyskinesis, andlastly painful symptoms of angina. Even though ischemic myocytesexperience physiological and metabolic changes within seconds of thecessation of coronary flow, resulting in T wave and sometimes ST segmentabnormalities (but without serum enzyme elevation), no cell deathresults from the ischemia [Kloner, R. A. and Jennings, R B, Circulation104: 2981-89 (2001)]. Once blood flow is re-established, a completerecovery of myocyte contractile function takes place.

Although angina pectoris (chest pain) may be a symptom of transientischemia, by and large transient ischemia is silent (meaning ST-segmentdepression of at least 1 mm is present without associated symptoms,e.g., chest pain) in 79% of subjects. In most patients with stableangina, for example, physical effort or emotion, with a resultantincrease in heart rate, blood pressure, or contractile state, or anycombination thereof, increases myocardial oxygen demand without anadequate delivery in oxygen delivery through tightly narrowed (stenosed)coronary arteries. More than 40% of patients with stable angina treatedwith one or more antianginal drugs have frequent episodes of silentischemia, which has been shown to predict a higher risk of coronaryevents and cardiac death [Deedwania, P C, Carbajal, E V, Arch. Intern.Med. 150: 2373-2382 (1991)].

Chronic Myocardial Ischemia

The term “chronic myocardial ischemia (CMI)” as used herein refers to aprolonged subacute or chronic state of myocardial ischemia due tonarrowing of a coronary blood vessel in which the myocardium“hibernates”, meaning that the myocardium downregulates or reduces itscontractility, and hence its myocardial oxygen demand, to match reducedperfusion, thereby preserving cellular viability and preventingmyocardial necrosis. This hibernating myocardium is capable of returningto normal or near-normal function on restoration of an adequate bloodsupply. Once coronary blood flow has been restored to normal or nearnormal and ischemia is resolved, however, the hibernating myocardiumstill does not contract. This flow-function mismatch resulting in a slowreturn of cardiac function after resolution of ischemia has been calledstunning. The length of time for function to return is quite variable,ranging from days to months, and is dependent on a number of parameters,including the duration of the original ischemic insult, the severity ofischemia during the original insult, and the adequacy of the return ofthe arterial flow. A number of studies have provided evidence forinflammation in hibernating myocardium [Heusch, G. et al., Am. J.Physiol. Heart Circ. Physiol. 288: 984-99 (2005)]. A study conducted ina porcine model of myocardial hibernation in which the mean rest (leftanterior descending coronary artery (LAD) coronary blood flow wasreduced to about 60% of baseline for a period of 24 hours to four weeks,detected apoptotic myocytes in all experimental pigs in the hibernatingregions supplied by the stenotic LAD, suggesting that functionaldownregulation may not be adequate to prevent gradual, ongoing myocytedeath through apoptosis in hibernating myocardium [Chen, C, et al., J.Am. Coll. Cardiol. 30: 1407-12 (1997)].

Acute Myocardial Infarction (AMI)

Another type of insult occurs during AMI. AMI is an abrupt change in thelumen of a coronary blood vessel that results in ischemic infarction,meaning that it continues until heart muscle dies. On gross inspection,myocardial infarction can be divided into two major types: transmuralinfarcts, in which the myocardial necrosis involves the full or nearlyfull thickness of the ventricular wall, and subendocardial(non-transmural) infarcts, in which the myocardial necrosis involves thesubendocardium, the intramural myocardium, or both, without extendingall the way through the ventricular wall to the epicardium. There oftenis total occlusion of the vessel with ST segment elevation because ofthrombus formation within the lumen as a result of plaque rupture. Theprolonged ischemic insult results in apoptotic and necroticcardiomyocyte cell death [See Kajstura, J., et al., Lab Invest. 74:86-107 (1996)]. Necrosis compromises the integrity of the sarcolemmalmembrane and intracellular macromolecules such that serum cardiacmarkers, such as cardiac-specific troponins and enzymes, such as serumcreatine kinase (CK), are released. In addition, the patient may haveelectrocardiogram (ECG) changes because of full thickness damage to themuscle. An ST-Elevation Myocardial Infarction (STEMI) is a larger injurythan a non-ST-elevation myocardial infarction. ST-segment elevation andQ waves on the ECG, two features highly indicative of myocardialinfarction, are seen in only about half of myocardial infarction caseson presentation.

AMI remains common with a reported annual incidence of 1.1 million casesin the United States alone [Antman, E. M., Braunwald, E., AcuteMyocardial Infarction, in Principles of Internal Medicine, 15th Ed.,Braunwald, E. et al., Eds., New York: McGraw-Hill (2001)]. Preclinicaland clinical data demonstrate that following a myocardial infarction,the acute loss of myocardial muscle cells and the accompanyingperi-infarct border zone hypo-perfusion result in a cascade of eventscausing an immediate diminution of cardiac function, with the potentialfor long term persistence. The extent of myocardial cell loss isdependent on the duration of coronary artery occlusion, existingcollateral coronary circulation and the condition of the cardiacmicrovasculature [Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer,M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheilban, I. e.al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M.R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13(1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002); Hirai, T.et al., Circulation 79: 791-96 (1989); Ejiri, M. et al., J. Cardiology20: 31-37 (1990)]. Because myocardial cells have virtually no ability toregenerate, myocardial infarction leads to permanent cardiac dysfunctiondue to contractile-muscle cell loss and replacement with nonfunctioningfibrotic scarring [Frangogiannis, N. G., et al., Cardiovascular Res.53(1): 31-47 (2002)]. Moreover, compensatory hypertrophy of viablecardiac muscle leads to microvascular insufficiency, which results infurther demise in cardiac function by causing myocardial musclehibernation and apoptosis of hypertrophied myocytes in the peri-infarctborder zone.

Among survivors of myocardial infarction, residual cardiac function isinfluenced by the extent of ventricular remodeling (meaning changes insize, shape, and function, typically a progressive decline in function,of the heart after injury). Alterations in ventricular topography(meaning the shape, configuration, or morphology of a ventricle) occurin both infarcted and healthy cardiac tissue after myocardial infarction[Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990)].Ventricular dilatation (meaning a stretching, enlarging or spreading outof the ventricle) causes a decrease in global cardiac function and isaffected by the infarct size, infarct healing and ventricular wallstresses. Recent efforts to minimize remodeling have been successful bylimiting infarct size through rapid reperfusion (meaning restoration ofblood flow) using thromobolytic agents, and mechanical interventions,including, but not limited to, placement of a stent, along with reducingventricular wall stresses by judicious use of pre-load therapies andproper after-load management [Pfeffer, M. A., Braunwald, E., Circulation81: 1161-72 (1990)]. Regardless of these interventions, a substantialpercentage of patients experience clinically relevant and long-termcardiac dysfunction after myocardial infarction [Sheiban, I. et al., J.Am. Coll. Cardiol. 38: 464-71 (2001)]. Despite revascularization of theinfarct related artery circulation and appropriate medical management tominimize ventricular wall stresses, a significant percentage of thesepatients experience ventricular remodeling, permanent cardiacdysfunction, and consequently remain at an increased lifetime risk ofexperiencing adverse cardiac events, including death [Paul et al., Am.Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation81: 1161-72 (1990)].

At the cellular level, immediately following a myocardial infarction,transient generalized cardiac dysfunction uniformly occurs. In thesetting of a brief (i.e., lasting three minutes to five minutes)coronary artery occlusion, energy metabolism is impaired, leading todemonstrable cardiac muscle dysfunction that can persist for up to 48hours despite immediate reperfusion. This so-called “stunned myocardiumphenomenon” occurs subsequent to or after reperfusion and is thought tobe a result of reactive oxygen species. The process is transient and isnot associated with an inflammatory response [Frangogiannis, N. G., etal., Cardiovascular Res. 53(1): 31-47 (2002)]. After successfulrevascularization, significant recovery from stunning occurs withinthree to four days, although complete recovery may take much longer[Boli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata,K. et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al.,Lancet 364: 141-48 (2004)]. Inflammation

Coronary artery occlusion of more significant duration, i.e., lastingmore than five minutes, leads to myocardial ischemia (i.e. aninsufficient blood flow to the heart's muscle mass) and is associatedwith a significant inflammatory response that begins immediately afterreperfusion and can last for up to several weeks [Frangogiannis, N. G.,et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. etal., Circulation 98: 687-798 (1998)]. The inflammatory process followingreperfusion is complex. Initially it contributes to myocardial damagebut later leads to healing and scar formation. This complex processappears to occur in two phases. In the first so-called “hot” phase(within the first five days), reactive oxygen species (in the ischemicmyocardial tissue) and complement activation generate a signalchemotactic for leukocytes (chemotaxis is the directed motion of amotile cell, organism or part towards environmental conditions it deemsattractive and/or away from surroundings it finds repellent) andinitiate a cytokine cascade [Lefer, D. J., Granger, D. N., Am. J. Med.4:315-23 (2000); Frangogiannis, N. G., et al., Circulation 7:699-710(1998)]. Mast cell degranulation, tumor necrosis factor alpha (TNFα)release, and increased interleukin-6 (IL-6), intercellular adhesionmolecule 1 (“ICAM-1” or CD-54, a receptor typically expressed onendothelial cells and cells of the immune system), selectin (L, E and P)and integrin (CD11a, CD11b and CD18) expression all appear to contributeto neutrophil accumulation and degranulation in ischemic myocardium[Frangogiannis, N. G. et al., Circulation 7: 699-710 (1998),Kurrelmeyer, K. M, et al., Proc. Natl Acad. Sci USA. 10: 5456-61 (2000);Lasky, L. A., Science 258: 964-69 (1992); Ma, X. L., et al., Circulation88(2): 649-58 (1993); Simpson, P. J. et al., J. Clin. Invest. 2: 624-29(1998)]. Neutrophils contribute significantly to myocardial cell damageand death through microvascular obstruction and activation of neutrophilrespiratory burst pathways after ligand-specific adhesion to cardiacmyocytes [Entman, M. L., et al., J. Clin. Invest. 4: 1335-45 (1992)].During the “hot” phase, angiogenesis is inhibited due to the release ofangiostatic substances, including interferon gamma-inducible protein (IP10) [Frangogiannis, N. G., et al., FASEB J. 15: 1428-30 (2001)].

In the second phase, the cardiac repair process begins (about day 6 toabout day 14), which eventually leads to scar formation (about day 14 toabout day 21) and subsequent ventricular remodeling (about day 21 toabout day 90). Soon after reperfusion, monocytes infiltrate theinfarcted myocardium. Attracted by complement (C5a), transforming growthfactor B 1 (“TGF-β1”) and monocyte chemotactic protein 1 (“MCP-1”),monocytes differentiate into macrophages that initiate the healingprocess by scavenging dead tissue, regulating extracellular matrixmetabolism, and inducing fibroblast proliferation [Birdshall, H. H., etal., Circulation 3: 684-92 (1997)]. Secretion of interleukin 10 (IL-10)by infiltrating lymphocytes also promotes healing by down-regulatinginflammatory cytokines and influencing tissue remodeling [Frangogiannis,N. G. et al., J. Immunol. 5:2798-2808 (2000)]. Mast cells also appear tobe involved in the later stages of myocardial repair by participating inthe formation of fibrotic scar tissue. Stem Cell Factor (SCF) is apotent attractor of mast cells. SCF mRNA has been shown to beup-regulated in ischemic myocardial segments in a canine model ofmyocardial infarction and thus may contribute to mast cell accumulationat ischemic myocardial sites [Franigogiannis, N. G. et al., Circulation98: 687-798 (1998)]. Mast cell products (including TGF-β, basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) and gelatinases A and B) induce fibroblast proliferation,influence extracellular matrix metabolism, and induce angiogenesis[Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., etal., Cardiology 93: 168-74 (2000)].

Initiation of the Inflammatory Process

Complement Activation

Hill and Ward [Hill J H, Ward P A. The phlogistic role of C3 leukotacticfragment in myocardial infarcts of rats. J Exp Med 1971; 885-890] werethe first to demonstrate that ischemic myocardial injury can activatethe complement cascade in a rat model of myocardial infarction.Subsequently, Pinckard et al. [Pinckard R. N., Olson M. S., Giclas P.C., et al. Consumption of classical complement components by heartsubcellular membranes in vitro and in patients after acute myocardialinfarction. J Clin Invest 1975; 3:740-750] suggested that myocardialcell necrosis results in the release of subcellular membraneconstituents rich in mitochondria, which are capable of triggering theearly acting components (C1, C4, C2 and C3) of the complement cascade.Rossen et al. [Rossen R. D., Michael L. H., Hawkins H. K., et al.Cardiolipin-protein complexes and initiation of complement activationafter coronary artery occlusion. Circ Res 1994; 3:546-555] havesuggested that during myocardial ischemia, mitochondria, extrudedthrough breaks in the sarcolemma, unfold and release membrane fragmentsrich in cardiolipin and protein. By binding C1 and supplying sites forthe assembly of later acting complement components, these subcellularfragments provide the means to disseminate the complement-mediatedinflammatory response to ischemic injury. mRNA and proteins for all thecomponents of the classical complement pathway are up-regulated in areasof myocardial infarcts [Vakeva A. P., Agah A., Rollins S. A., et al.Myocardial infarction and apoptosis after myocardial ischemia andreperfusion: role of the terminal complement components and inhibitionby anti-C5 therapy. Circulation 1998; 22:2259-2267; Yasojima K., KilgoreK. S., Washington R. A., Lucchesi B. R., McGeer P. L. Complement geneexpression by rabbit heart: up-regulation by ischemia and reperfusion.Circ Res 1998; 11:1224-1230].

Complement activation may play an important role in mediating neutrophiland monocyte recruitment in the injured myocardium. Dreyer et al.[Dreyer W. J., Michael L. H., Nguyen T., et al. Kinetics of C5a releasein cardiac lymph of dogs experiencing coronary arteryischemia-reperfusion injury. Circ Res 1992; 6:1518-1524] showed thatpost-ischemic cardiac lymph contains leukocyte chemotactic activity,which is maximal during the first hour of reperfusion with washoutwithin the next 3 hours.

Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS) are molecules with unpaired electrons intheir outer orbit. They have the potential to directly injure cardiacmyocytes and vascular cells and may be involved in triggeringinflammatory cascades through the induction of cytokines [Lefer D. J.,Granger D. N. Oxidative stress and cardiac disease. Am J Med 2000;4:315-323; Dhalla N. S., Elmoselhi A. B., Hata T., Makino N. Status ofmyocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res2000; 3:446-456]. Reactive oxygen species have been shown to exert adirect inhibitory effect on myocardial function in vivo and have acritical role in the pathogenesis of myocardial stunning [Bolli R.Oxygen-derived free radicals and postischemic myocardial dysfunction(‘stunned myocardium’). J Am Coll Cardiol 1988; 1:239-249]. In addition,evidence exists for a potential role of reactive oxygen in leukocytechemotaxis [Granger D. N. Role of xanthine oxidase and granulocytes inischemia-reperfusion injury. Am J Physiol 1988; 6(2):H1269-H1275].Potential mechanisms through which reactive oxygen intermediates maygenerate a leukotactic stimulus include complement activation, inductionof P-selectin expression, chemokine upregulation, and an increase in theability of endothelial ICAM-1 to bind to neutrophils [Shingu M.,Nobunaga M. Chemotactic activity generated in human serum from the fifthcomponent of complement by hydrogen peroxide. Am J Pathol 1984;2:201-206; Akgur F. M., Brown M. F., Zibari G. B., et al. Role ofsuperoxide in hemorrhagic shock-induced P-selectin expression. Am JPhysiol Heart Circ Physiol 2000; 2:H791-H797; Patel K. D., Zimmerman G.A., Prescott S. M., McEver R. P., McIntyre T. M. Oxygen radicals inducehuman endothelial cells to express GMP-140 and bind neutrophils. J CellBiol 1991; 4:749-759; Lakshminarayanan V., Drab-Weiss E. A., Roebuck K.A. H₂O₂ and tumor necrosis factor-alpha induce differential binding ofthe redox-responsive transcription factors AP-1 and NF-kappa B to theinterleukin-8 promoter in endothelial and epithelial cells. J Biol Chem1998; 49:32670-32678; Lakshminarayanan V., Beno D. W., Costa R. H.,Roebuck K. A. Differential regulation of interleukin-8 and intercellularadhesion molecule-1 by H₂O₂ and tumor necrosis factor-alpha inendothelial and epithelial cells. J Biol Chem 1997; 52:32910-32918;Sellak H., Franzini E., Hakim J., Pasquier C. Reactive oxygen speciesrapidly increase endothelial ICAM-1 ability to bind neutrophils withoutdetectable up-regulation. Blood 1994; 9:2669-2677].

Most of the evidence implicating ROS in the pathophysiology ofmyocardial infarction is derived from investigations using free radicalscavengers. Jolly et al. [Jolly S. R., Kane W. J., Bailie M. B., AbramsG. D., Lucchesi B. R. Canine myocardial reperfusion injury. Itsreduction by the combined administration of superoxide dismutase andcatalase. Circ Res 1984; 3:277-285] demonstrated that the combination ofthe antioxidant enzymes superoxide dismutase and catalase significantlyreduced infarct size in dogs undergoing experimental myocardial ischemiaand reperfusion. Other investigators found similar beneficial effects ofantioxidant interventions in experimental models of myocardialinfarction. However, there is a significant number of studies describinga failure of antioxidants to prevent injury or demonstrating an earlyprotective effect, which waned with increased duration of reperfusion[Uraizee A., Reimer K. A., Murry C. E., Jennings R. B. Failure ofsuperoxide dismutase to limit size of myocardial infarction after 40minutes of ischemia and 4 days of reperfusion in dogs. Circulation 1987;6:1237-1248; Gallagher K. P., Buda A. J., Pace D., Gerren R. A., ShlaferM. Failure of superoxide dismutase and catalase to alter size ofinfarction in conscious dogs after 3 hours of occlusion followed byreperfusion. Circulation 1986; 5:1065-1076; Richard V. J., Murry C. E.,Jennings R. B., Reimer K. A. Therapy to reduce free radicals duringearly reperfusion does not limit the size of myocardial infarcts causedby 90 minutes of ischemia in dogs. Circulation 1988; 2:473-480].Recently, transgenic mice that overexpress superoxide dismutase (SOD1)showed significant protection from post-ischemic injury and asignificant decrease in infarct size in Langendorf-perfused heartsundergoing left coronary artery ligation [Wang P., Chen H., Qin H., etal. Overexpression of human copper, zinc-superoxide dismutase (SOD1)prevents post-ischemic injury. Proc Natl Acad Sci USA 1998; 8:4556-4560;Chen Z., Siu B., Ho Y. S., et al. Overexpression of MnSOD protectsagainst myocardial ischemia/reperfusion injury in transgenic mice. J MolCell Cardiol 1998; 11:2281-2289]. Unfortunately, two clinical studiesusing recombinant human superoxide dismutase in patients with acutemyocardial infarction undergoing thrombolysis [Murohara Y., Yui Y.,Hattori R., Kawai C. Effects of superoxide dismutase on reperfusionarrhythmias and left ventricular function in patients undergoingthrombolysis for anterior wall acute myocardial infarction. Am J Cardiol1991; 8:765-767.] or balloon angioplasty [Flaherty J. T., Pitt B.,Gruber J. W., et al. Recombinant human superoxide dismutase (h-SOD)fails to improve recovery of ventricular function in patients undergoingcoronary angioplasty for acute myocardial infarction. Circulation 1994;5:1982-1991] demonstrated no significant improvement in left ventricularfunction. In addition, prolonged coronary occlusion (>2 h) is usuallypresent in the clinical setting of reperfused myocardial infarction andmay cause extensive irreversible myocardial damage, leaving fewermyocytes to be affected by free radical-mediated injury [Lefer D. J.,Granger D. N. Oxidative stress and cardiac disease. Am J Med 2000;4:315-323; Maxwell S. R., Lip G. Y. Reperfusion injury: a review of thepathophysiology, clinical manifestations and therapeutic options. Int JCardiol 1997; 2:95-117].

Meldrum et al. [Meldrum D R, Dinarello C A, Cleveland J C, Jr, et al.Hydrogen peroxide induces tumor necrosis factor alpha mediated cardiacinjury by a p38 mitogen activated protein kinase dependent mechanisms.Surgery. 1998; 124:291-296. discussion 297] demonstrated that H₂O₂ aloneinduced myocardial TNF-α mediated cardiac injury by a p38mitogen-activated protein kinase (MAPK)-dependent mechanism. It has beenhypothesized that reactive oxygen intermediates may generate aleukotatic stimulus that includes, complement activation, induction ofhemorrhagic shock-induced P-selectin expression, chemokine up-regulationand an increase in the endothelial intercellular adhesion molecule(ICAM)-1 ability to bind neutrophils [Shingu M, Nobunaga M. Chemotacticactivity generated in human serum from the fifth component of complementby hydrogen peroxide. Am J Pathol. 1984; 117:201-206; Akgur F M, Brown MF, Zibari G B, et al. Role of superoxide in hemorrhagic shock-inducedP-selectin expression. Am J Physiol Heart Circ Physiol. 2000;279:H791-H797; Lakshminarayanan V, Beno D W, Costa R H, Roebuck Kans.Differential regulation of interleukin-8 and intercellular adhesionmolecule-1 by H₂O₂ and tumor necrosis factor-alpha in endothelial andepithelial cells. J Biol Chem. 1997; 272:32910-32918; Sellak H, FranziniE, Hakim J, Pasquier C. Reactive oxygen species rapidly increaseendothelial ICAM-1 ability to bind neutrophils without detectableup-regulation. Blood. 1994; 83:2669-2677]. It was reported that the useof the antioxidant enzymes superoxide dismutase and catalase reducedinfarct size in dogs with myocardial ischemia and reperfusion [Jolly SR, Kane W J, Bailie M B, Abrams G D, Lucchesi B R. Canine myocardialreperfusion injury: its reduction by the combined administration ofsuperoxide dismutase and catalase. Circ Res. 1984; 54:277-285]. However,failed studies have been reported where antioxidant treatment was usedto prevent myocardial ischemic injury [Uraizee A, Reimer K A, Murry C E,Jennings R B. Failure of superoxide dismutase to limit size ofmyocardial infarction after 40 minutes of ischemia and 4 days ofreperfusion in dogs. Circulation. 1987; 75:1237-1248; Gallagher K P,Buda A J, Pace D, Gerren R A, Shlafer M. Failure of superoxide dismutaseand catalase to alter size of infarction in conscious dogs after 3 hoursof occlusion followed by reperfusion. Circulation. 1986; 73:1065-1076].For example, two clinical studies in which recombinant human superoxidedismutase was used in patients with an acute myocardial infarctionundergoing percutaneous coronary intervention or thrombolysis showed nosignificant improvement of left ventricular function [Murohara Y, Yui Y,Hattori R, Kawai C. Effects of superoxide dismutase on reperfusionarrhythmias and left ventricular function in patients undergoingthrombolysis for anterior wall acute myocardial infarction. Am JCardiol. 1991; 67:765-767; Flaherty J T, Pitt B, Gruber J W, et al.Recombinant human superoxide dismutase (h-SOD) fails to improve recoveryof ventricular function in patients undergoing coronary angioplasty foracute myocardial infarction. Circulation. 1994; 89:1982-1991].

Cytokine Cascade

Experimental myocardial infarction is associated with the coordinatedactivation of a series of cytokine and adhesion molecule genes. Acritical element in the regulation of these genes involves the complexformed by NF-κB and Iκβ [Lenardo M. J., Baltimore D. NF-kappa B: apleiotropic mediator of inducible and tissue-specific gene control. Cell1989; 2: 227-229]. NF—KB is activated by a vast number of agents,including cytokines (such as TNF-α and IL-1β) and free radicals.Cytokines can self-amplify through a positive feedback loop targetingthe nuclear factor (NF)-κB. Up-regulation of TNF-α in the infarctmyocardium can up-regulate the levels of TNF-α in the neighboring normalmyocardium, leading to amplified cytokine effects [Irwin M, Mak S, MannD L, et al. Tissue expression and immunolocalization of tumor necrosisfactor-alpha in post infarction-dysfunctional myocardium. Circulation.1999; 99:1492-1498]. TNF-α stimulates expression of pro-inflammatorycytokines, chemokines and adhesion molecules by leukocytes andendothelial cells, and regulates extracellular matrix metabolism byreducing collagen synthesis and by enhancing matrix metalloprotease(MMP) activity in cardiac fibroblasts; other adhesive cytokines, such asmonocyte chemoattractant protein (MCP)-1, are also induced in theischemic and re-perfused canine myocardium [Siwik D A, Chang D L, ColuciW S. Interleukin-1 beta and tumor necrosis factor-alpha decreasecollagen synthesis and increase matrix metalloproteinase activity incardiac fibroblasts in vitro. Circ Res. 2000; 86:1259-1265]. Kumar etal. [Kumar A G, Ballantyne C M, Michael L H, et al. Induction ofmonocyte chemoattractant protein-1 in the small veins of the ischemicand re-perfused canine myocardium. Circulation. 1997; 95:693-700]suggested that MCP-1 plays a significant role in monocyte trafficking inre-perfused myocardium.

The mechanisms responsible for triggering the cytokine cascade in theinfarcted myocardium have only recently been investigated. Severalstudies have indicated a role for preformed mast cell-derived mediatorsin initiating the cytokine cascade ultimately responsible for ICAM-1induction in the re-perfused canine myocardium [Frangogiannis N. G.,Lindsey M. L., Michael L. H., et al. Resident cardiac mast cellsde-granulate and release preformed TNF-alpha, initiating the cytokinecascade in experimental canine myocardial ischemia/reperfusion.Circulation 1998; 7:699-710; Frangogiannis N. G., Entman M. L. Mastcells in myocardial ischemia and reperfusion, Mast cells and basophilsin physiology, pathology and host defense. In: Marone G., LiechtensteinL. M., Galli S. J., editors. London: Academic Press; 2000. p. 507-522;Frangogiannis N. G., Burns A. R., Michael L. H., Entman M. L.Histochemical and morphological characteristics of canine cardiac mastcells. Histochem J 1999; 4:221-229]. Mast cells have been recognized asan important source of preformed and newly synthesized cytokines,chemokines and growth factors [Frangogiannis N. G., Lindsey M. L.,Michael L. H., et al. Resident cardiac mast cells de-granulate andrelease preformed TNF-alpha, initiating the cytokine cascade inexperimental canine myocardial ischemia/reperfusion. Circulation 1998;7:699-710; Frangogiannis N. G., Entman M. L. Mast cells in myocardialischaemia and reperfusion, Mast cells and basophils in physiology,pathology and host defense. In: Marone G., Liechtenstein L. M., Galli S.J., editors. London: Academic Press; 2000. p. 507-522; Frangogiannis N.G., Burns A. R., Michael L. H., Entman M. L. Histochemical andmorphological characteristics of canine cardiac mast cells. Histochem J1999; 4:221-229]. Gordon and Galli [Gordon J R, Galli S J. Mast cells asa source of both preformed and immunologically inducibleTNF-alpha/cachectin. Nature 1990; 274-276; Gordon J. R., Burd P. R.,Galli S. J. Mast cells as a source of multifunctional cytokines. ImmunolToday 1990; 12:458-464] identified mouse peritoneal mast cells as animportant source of both preformed and immunologically-induced TNF-α.The constitutive presence of TNF-α in canine cardiac mast cells have ledto the hypothesis that mast cell-derived TNF-α may be released followingmyocardial ischemia, representing an ‘upstream’ cytokine responsible forinitiating the inflammatory cascade [Frangogiannis N G, CardiovascularResearch (2002) Vol. 53, Issue 1, pp. 31-47].

Moreover, it has been reported that early post-ischemic cardiac lymph iscapable of inducing IL-6 expression in canine mononuclear cells invitro. Incubation with a neutralizing antibody to TNF-α in partinhibited IL-6 up-regulation, suggesting an important role for TNF-α asthe upstream cytokine inducer. Mast cell degranulation appears to beconfined in the ischemic area and results in rapid release of TNF-α,inducing IL-6 in infiltrating mononuclear cells [Frangogiannis N G,Cardiovascular Research (2002) Vol. 53, Issue 1, pp. 31-47; 56,61].

The role of TNF-α in myocardial infarction is thought to be more complexthan simply serving as a trigger of a cytokine cascade [Sack M. N.,Smith R. M., Opie L. H. Tumor necrosis factor in myocardial hypertrophyand ischemia—an anti-apoptotic perspective. Cardiovasc Res 2000;3:688-695; Belosjorow S., Schulz R., Dorge H., Schade F. U., Heusch G.Endotoxin and ischemic preconditioning: TNF-alpha concentration andmyocardial infarct development in rabbits. Am J Physiol 1999;6(2):H2470-H2475]. Recent experiments using TNFR1/TNFR2 double receptorknockout mice undergoing left coronary artery ligation had significantlylarger infarct size and increased myocyte apoptosis when compared withwild-type controls [Kurrelmeyer K. M., Michael L. H., Baumgarten G., etal. Endogenous tumor necrosis factor protects the adult cardiac myocyteagainst ischemic-induced apoptosis in a murine model of acute myocardialinfarction. Proc Natl Acad Sci USA 2000; 10:5456-5461]. These findingssuggested that TNF-α may induce a cytoprotective signal capable ofpreventing or delaying the development of myocyte apoptosis followingmyocardial infarction.

Other studies have shown that TNF-α expression during the healing phasewas not confined to the infarct or peri-infarct zone, but was alsolocalized in the normal non-infarcted myocardium, in which remodelingwas ongoing. Thus, sustained TNF-α expression may have a role in thereparative process following myocardial infarction [Irwin M. W., Mak S.,Mann D. L., et al. Tissue expression and immunolocalization of tumornecrosis factor-alpha in post-infarction dysfunctional myocardium.Circulation 1999; 11:1492-1498; Jacobs M., Staufenberger S., Gergs U.,et al. Tumor necrosis factor-alpha at acute myocardial infarction inrats and effects on cardiac fibroblasts. J Mol Cell Cardiol 1999;11:1949-1959].

Cytokine and Chemokine Upregulation

Chemokine up-regulation is a noted feature of the post-infarctioninflammatory response (Table 1) [Frangogiannis N G. Chemokines inischemia and reperfusion. Thromb Haemost. 2007; 97:738-747].Investigators have demonstrated strong induction of several chemokinesin the ischemic myocardium, supporting their role in leukocyterecruitment [Birdsall H H, Green D M, Trial J, et al. Complement C5a,TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes intoischemic canine myocardium within the first one to five hours afterreperfusion. Circulation. 1997; 95:684-692]. MCP-1 up-regulation hasbeen demonstrated in a mouse model [Tarzami S T, Cheng R, Miao W, KitsisR N, Berman J W. Chemokine expression in myocardial ischemia: MIP-2dependent MCP-1 expression protects cardiomyocytes from cell death. JMol Cell Cardiol. 2002; 34:209-221]. Frangogiannis reported that aMCP-1−/− infarct mouse model had decreased messenger ribonucleic acid(mRNA) expression of the cytokines TNF-α, IL-1β, TGF-β and IL-10, andshowed defective macrophage differentiation [Frangogiannis N G.Chemokines in ischemia and reperfusion. Thromb Haemost. 2007;97:738-747]. Cytokines, such as TNF-α and IL-6, are rapidly released inthe central zone during a myocardial infarction; however, they areusually maximal in the border zone [Irwin M, Mak S, Mann D L, et al.Tissue expression and immunolocalization of tumor necrosis factor-alphain post infarction-dysfunctional myocardium. Circulation. 1999;99:1492-1498; Gwechenberger M, Mendoza L H, Youker K A, et al. Cardiacmyocytes produce interleukin-6 in culture and in viable border zone ofre-perfused infarctions. Circulation. 1999; 99:546-551]. This robustup-regulation may return to baseline levels if the infarction is small;if the infarction is large and the inflammatory response is excessive,there can be sustained cytokine up-regulation, corresponding to achronic remodeling phase.

TABLE 1 Up-regulated chemokines and their role after myocardial ischemiaand reperfusion. Action After Myocardial Ischemia Chemokine andReperfusion CXCL8/Interleukin (IL)-8 Induce neutrophil infiltrationCCL2/Monocyte Chemoattractant Regulate monocyte and lymphocyte Protein(MCP)-1 recruitment CCL3/Macrophage Inflammatory Regulate monocyte andlymphocyte Protein (MIP)-1α recruitment CCL4/Macrophage InflammatoryRegulate monocyte and lymphocyte Protein (MIP)-1β recruitmentCXCL10/Interferon-10 Angiostatic factor with anti-fibrotic properties[taken from Nah D-Y, Rhee M-Y, Korean Circ. J. October 2009; 39(10):393-398]

Cell-Mediated Inflammatory Response to Myocardial Infarction

Neutrophils

Neutrophils are recruited during the initial stage of cardiac ischemicinjury. Neutrophil transmigration in the infarcted myocardium requiresadhesive interactions with activated vascular endothelial cells.Neutrophils may secrete oxidants and proteases and possibly expressmediators capable of amplifying cell recruitment [Frangogiannis N G,Youker K A, Entman M L. The role of the neutrophil in myocardialischemia and reperfusion. EXS. 1996; 76:263-284]. Neutrophil depletionin animals undergoing re-perfused myocardial infarction has beenreported to significantly decrease the infarct size, suggesting that asignificant amount of myocardial injury may be induced by neutrophildependent mechanisms [Romson J L, Hook B G, Kunkel S L, Abrams G D,Schork M A, Lucchesi B R. Reduction of the extent of ischemic myocardialinjury by neutrophil depletion in the dog. Circulation. 1983;67:1016-1023; Jordan J E, Zhao Z Q, Vinten-Johansen J. The role ofneutrophils in myocardial ischemia-reperfusion injury. Cardiovasc Res.1999; 43:860-878].

The mechanisms associated with neutrophil-induced myocardial ischemicinjury have not been identified. Jaeschke et al. [Jaeschke H, Smith C W.Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol.1997; 61:647-653] suggested that neutrophils may directly injureparenchymal cells through release of specific toxic products. Whileselectins have been implicated, there have been inconsistent results ofselectin-related interventions in experimental models of myocardialischemia [Jones S P, Girod W G, Granger D N, Palazzo A J, Lefer D J.Reperfusion injury is not affected by blockade of P-selectin in thediabetic mouse heart. Am J Physiol. 1999; 277:H763-H769; Birnbaum Y,Patterson M, Kloner R A. The effect of CY1503, a sialyl Lewis X analogblocker of the selectin adhesion molecules, on infarct size and “noreflow” in the rabbit model of acute myocardial infarction/reperfusion.J Mol Cell Cardiol. 1997; 29:2013-2025]. The selectin family consists ofL-selectin, P-selectin and E-selectin. P-selectin expression occursrapidly in endothelial cells during cardiac ischemic injury.Experimental studies have suggested that monoclonal antibodies againstP-selectin reduced myocardial necrosis, preserving coronary endothelialfunction and attenuating neutrophil infiltration in ischemic andreperfused myocardium [Palazzo A J, Jones S P, Anderson D C, Granger DN, Lefer D J. Coronary endothelial P-selectin in pathogenesis ofmyocardial ischemia-reperfusion injury. Am J Physiol. 1998;275:H1865-H1872].

Mononuclear Cells

MCP-1/CCL2 plays an important role in monocyte recruitment to theinfarcted myocardium [Dewald O, Zymek P, Winkelmann K, et al.CCL2/monocyte chemoattractant protein-1 regulates inflammatory responsescritical to healing myocardial infarcts. Circ Res. 2005; 96:881-889].Cytokines, such as TGF-β, free radical oxygen, complement, and the CCchemokines (e.g., MCP-1) may also play a role in monocyte infiltration.Infiltration of monocytes into the infarcted myocardium is followed bymaturation and differentiation of these blood-derived cells intomacrophages.

Cardiac Repair After Myocardial Infarction

TGF-β as a Key Regulator in Cardiac Repair

TGF-β is a multifunctional cytokine that controls proliferation andcellular differentiation in most cells. The exact role of TGF-βsignaling in the infarcted and remodeled heart is poorly understood. Itsrole in myocardial infarction is thought to involve cardiomyocytehypertrophy, angiogenic or angiostatic effects, reduced adhesionmolecule expression, macrophage deactivation, chemokine and cytokinerepression, myofibroblast differentiation, fibroblast proliferation andextracellular matrix protein synthesis [Nah D-Y, Rhee M-Y, Korean Circ.J. October 2009; 39(10): 393-39847; Frangogiannis N G. The immune systemand cardiac repair. Pharmacol Res. 2008; 58:88-111].

TGF-β was shown to be significantly up-regulated and TGF-β mRNA andprotein was significantly increased at the infarct border zone in anexperimental rat model of myocardial infarction [Thompson N L, BazoberryF, Speir E H, et al. Transforming growth factor beta-1 in acutemyocardial infarction in rats. Growth Factors. 1988; 1:91-99; Dean R G,Balding L C, Candido R, et al. Connective tissue growth factor andcardiac fibrosis after myocardial infarction. J Histochem Cytochem.2005; 53:1245-1256]. During infarct healing, TGF-β may play a role inthe suppression of chemokine and cytokine synthesis and is thought to bea key mediator of the transition from inflammation to fibrosis [BassolsA, Massague J. TGF-β regulates the expression and structure ofextracellular matrix chondroitin/dermatan sulfate proteoglycans. J BiolChem. 1988; 263:3039-3045]. Lefer et al. [Lefer A M, Tsao P, Aoki N,Palladino M A., Jr Mediation of cardio-protection by transforming growthfactor-beta. Science. 1990; 249:61-64] reported that TGF-β injectionsreduced myocardial ischemic injury mediated by pro-inflammatorycytokines such as TNF-α during the inflammatory phase of myocardialhealing. Anti-TGF-β treatment before or after coronary artery ligationincreased mortality and worsened the left ventricular remodeling in micewith non-re-perfused myocardial infarction [Frantz S, Hu K, Adammek A,et al. Transforming growth factor beta inhibition increases mortalityand left ventricular dilatation after myocardial infarction. Basic ResCardiol. 2008; 103:485-492]. The inhibition of TGF-β signaling byinjection of a TGF-β II receptor resulted in reduction of leftventricular remodeling by modulation of cardiac fibrosis; early TGF-3inhibition increased mortality and left ventricular dilatation [IkeuchiM, Tsutsui H, Shiomi T, et al. Inhibition of TGF-beta signalingexacerbates early cardiac dysfunction but prevents late remodeling afterinfarction. Cardiovasc Res. 2004; 64:526-535; Okada H, Takemura G, KosaiK, et al., Postinfarction gene therapy against transforming growthfactor-beta signal modulates infarct tissue dynamics and attenuates leftventricular remodeling and heart failure. Circulation. 2005;111:2430-2437]. Youn et al. [Youn T J, Kim H S, Oh B H. Ventricularremodeling and transforming growth factor-beta 1 mRNA expression afternontransmural myocardial infarction in rats: effects of angiotensinconverting enzyme inhibition and angiotensin II type 1 receptorblockade. Basic Res Cardiol. 1999; 94:246-253] reported that anangiotensin converting enzyme inhibitor and angiotensin receptorblockade resulted in decreased TGF-β mRNA expression afternon-transmural infarction in the rat.

Other Cytokines in Cardiac Repair

Three IL-1 molecules (IL-1α, IL-β and IL-1 Ra) that are specificreceptor antagonists [Dinarello C A. Biologic basis for interleukin-1 indisease. Blood. 1996; 87:2095-2147] have been implicated in cardiacrepair. Bujak et al. [Bujak M, Dobaczewski M, Chatila K, et al.Interleukin-1 receptor type I signaling critically regulates infarcthealing and cardiac remodeling. Am J Pathol. 2008; 173:57-67]demonstrated that IL-1 signaling is essential for activation ofinflammatory and fibrogenic pathways in the healing infarct and plays animportant role in the pathogenesis of remodeling after infarction.

IL-10 exerts potent anti-inflammatory effects and modulates MMPexpression [de Waal Malefyt R, Abrams J, Bennett B, Figdor C G, de VriesJ E. Interleukin 10 (IL-10) inhibits cytokine synthesis by humanmonocytes: an auto-regulatory role of IL-10 produced by monocytes. J ExpMed. 1991; 174:1209-1220; Moore K W, deWaal Malefyt R, Coffman R L,O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu RevImmunol. 2001; 19:683-765; Lacraz S, Nicod L P, Chicheportiche R, WelgusH G, Dayer J M. IL-10 inhibits metalloproteinase and stimulates TIMP-1production in human mononuclear phagocytes. J Clin Invest. 1995;96:2304-2310]. However, Zymek et al. [Zymek P, Nah D Y, Bujak M, et al.Interleukin-10 is not a critical regulator of infarct healing and leftventricular remodeling. Cardiovasc. Res. 2007; 74:313-322] reported thatIL-10 signaling plays a noncritical role in the suppression ofinflammatory mediators, resolution of the inflammatory response andfibrous tissue deposition following myocardial infarction in the mouse;which may be due to the involvement of multiple overlapping regulatorymechanisms controlling various pro-inflammatory pathways activated inthe infarcted myocardium.

Proteins in Cardiac Repair

Cluster of differentiation 44 (CD44) is a cell surface glycoproteininvolved in cell-cell interaction and cell adhesion and migration.CD44-hyaluronan interactions play a role in leukocyte extravasation atthe inflammatory site and serves as a key factor in the resolution ofinflammation through removal of matrix breakdown products and clearanceof apoptotic neutrophils [Mikecz K, Brennan F R, Kim J H, Glant T T.Anti-CD44 treatment abrogates tissue edema and leukocyte infiltration inmurine arthritis. Nat Med. 1995; 1:558-563; DeGrendele H C, Estess P,Siegelman M H. Requirement for CD44 in activated T cell extravasationinto an inflammatory site. Science. 1997; 278:672-675; Teder P,Vandivier R W, Jiang D, et al. Resolution of lung inflammation by CD44.Science. 2002; 296:155-158]. Huebener et al. [Huebener P, Abou-Khamis T,Zymek P, et al. CD44 is critically involved in infarct healing byregulating the inflammatory and fibrotic response. J Immunol. 2008;180:2625-2633] tested the role of CD44 in infarct healing anddemonstrated that CD44 mRNA levels were significantly induced in theinfarcted heart; CD44 null mice showed enhanced and prolongedinflammation in the infarcted heart followed by decreased myofibroblastinfiltration, reduced collagen deposition and diminished proliferativeactivity. Huebener et al. concluded that CD44 is critically involved ininfarct healing by regulating the inflammatory and fibrotic response.

Thrombospondin (TSP)-1 is a TGF-β activator as well as an adhesiveglycoprotein involved in cell-to-cell and cell-to-matrix interactionwith potent angiostatic properties [Lawler J. Thrombospondin-1 as anendogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med.2002; 6:1-12]. TSP-1 showed selective localization in the infarct borderzone, and TSP-1 knockout animals had markedly increased macrophage andmyofibroblast density in the infarct and in remodeling of non-infarctedmyocardial areas, and was more extensive in post-infarction remodelingthan in wild-type mice. Frangogiannis et al. [Frangogiannis N G, Ren G,Dewald O, et al. The critical role of endogenous thrombospondin (TSP)-1in preventing expansion of healing myocardial infarcts. Circulation.2005; 111:2935-2942] concluded that the selective endogenous expressionof TSP-1 at the infarct border zone may serve as a “barrier,” limitingexpansion of granulation tissue and protecting the non-infarctedmyocardium from fibrotic remodeling.

Smad is an essential protein component of the TGF-β pathway [Shi Y,Massague J. Mechanisms of TGF-beta signaling from cell membrane to thenucleus. Cell. 2003; 113:685-700]. Hao et al. [Hao J, Ju H, Zhao S,Junail A, Scammell-La Fleur T, Dixon I M. Elevation of expression ofSmads 2, 3, and 4, decorin and TGF-beta in the chronic phase ofmyocardial infarct scar healing. J Mol Cell Cardiol. 1999; 31:667-678]showed that TGF-β mRNA was significantly increased in the infarct scarcompared to viable myocardium, and that Cardiac Smad 2, 3 and 4 proteinswere significantly increased in the border and scar tissues whencompared to viable myocardium, suggesting that TGF-β/Smad signaling maybe involved in the remodeling of the infarct scar.

The reparative phase of healing involves activation of proteinases,which are critical for cell migration and extracellular matrixremodeling. Recent studies have demonstrated that deficiency ofurokinase-type plasminogen activator (uPA) protected mice undergoingleft coronary artery ligation against myocardial rupture [Heymans S.,Luttun A., Nuyens D., et al. Inhibition of plasminogen activators ormatrix metalloproteinases prevents cardiac rupture but impairstherapeutic angiogenesis and causes cardiac failure. Nat Med 1999;10:1135-1142]. However, uPA −/− mice also showed impaired scar formationand infarct neovascularization. Furthermore, plasminogen-deficient miceshowed a profound disturbance in healing, suggesting a crucial role forthe proteolytic system in regulating cardiac repair [Creemers E.,Cleutjens J., Smits J., et al. Disruption of the plasminogen gene inmice abolishes wound healing after myocardial infarction. Am J Pathol2000; 6:1865-1873].

Matrix metalloproteinase (MMP) expression is upregulated in theinfarcted myocardium and may have a prominent role in extracellularmatrix remodeling. Administration of MMP inhibitors and targeteddeletion of MMP-9 attenuated left ventricular enlargement in murinemyocardial infarction [Cleutjens J. P., Kandala J. C., Guarda E.,Guntaka R. V., Weber K. T. Regulation of collagen degradation in the ratmyocardium after infarction. J Mol Cell Cardiol 1995; 6:1281-1292; LuL., Gunja-Smith Z., Woessner J. F., et al. Matrix metalloproteinases andcollagen ultrastructure in moderate myocardial ischemia and reperfusionin vivo. Am J Physiol Heart Circ Physiol 2000; 2:H601-H609; Rohde L. E.,Ducharme A., Arroyo L. H., et al. Matrix metalloproteinase inhibitionattenuates early left ventricular enlargement after experimentalmyocardial infarction in mice. Circulation 1999; 23:3063-30701; DucharmeA., Frantz S., Aikawa M., et al. Targeted deletion of matrixmetalloproteinase-9 attenuates left ventricular enlargement and collagenaccumulation after experimental myocardial infarction. J Clin Invest2000; 1:55-62].

Cardiac Fibroblasts and Extracellular Matrix Remodeling

Cardiac Fibroblasts

In the healthy heart, 70% of the cells present are fibroblasts [JugduttB I. Ventricular remodeling after infarction and the extracellularcollagen matrix: when is enough enough? Circulation. 2003; 108:1395-403;Banerjee I, Fuseler J W, Price R L, Borg T K, Baudino TA. Determinationof cell types and numbers during cardiac development in the neonatal andadult rat and mouse. Am J. Physiol Heart Circul Physiol. 2007;293:H1883-91]. Fibroblasts are widely distributed connective tissuecells that are found in all vertebrate organisms. They are usuallydefined as cells of mesenchymal origin that produce a variety ofextracellular matrix (ECM) components, including multiple collagens, aswell as fibronectin [Eghbali-Webb M. Molecular Biology Intelligence UnitMolecular Biology of Collagen Matrix in the Heart. Austin, Ill.: Landes;1994; Kanekar S, Hirozanne T, Terracio L, Borg T K. Cardiac fibroblasts:form and function. Cardiovasc Pathol. 1998; 7: 127-133].Morphologically, fibroblasts are flat, spindle-shaped cells withmultiple processes emanating from the main cell body. Fibroblasts lack abasement membrane, a characteristic feature that separates them from theother permanent cell types of the heart, all of which do contain abasement membrane.

Fibroblasts produce extracellular matrix constituents needed to supportcell ingrowth. Willems et al. [Willems I. E., Havenith M. G., De Mey J.G., Daemen M. J. The alpha-smooth muscle actin-positive cells in healinghuman myocardial scars. Am J Pathol 1994; 4:868-875] previouslyidentified and characterized interstitial nonvascular α-smooth muscleactin (α-SMAc) positive cells, which were present in human myocardialscars 4-6 days after an infarction. These cells are phenotypicallymodulated fibroblasts, termed myofibroblasts, that developultra-structural and phenotypic characteristics of smooth muscle cellsand are the predominant source of collagen mRNA in healing myocardialinfarcts[Gabbiani G. Evolution and clinical implications of themyofibroblast concept. Cardiovasc Res 1998; 3:545-548]. Myofibroblastsare undifferentiated cells that may be capable of assuming a variety ofdifferent roles, such as extracellular matrix metabolism, neovesselformation and contractile activity [Cleutjens J. P., Verluyten M. J.,Smiths J. F., Daemen M. J. Collagen remodeling after myocardialinfarction in the rat heart. Am J Pathol 1995; 2:325-338; Serini G.,Gabbiani G. Mechanisms of myofibroblast activity and phenotypicmodulation. Exp Cell Res 1999; 2:273-283; Cleutjens J. P., BlankesteijnW. M., Daemen M. J., Smits J. F. The infarcted myocardium: simply deadtissue, or a lively target for therapeutic interventions. Cardiovasc Res1999; 2:232-241]. TGF-β appears to play an important role inmyofibroblast differentiation during wound healing by regulating α-SMAcexpression in these cells [Desmouliere A., Geinoz A., Gabbiani F.,Gabbiani G. Transforming growth factor-beta 1 induces alpha-smoothmuscle actin expression in granulation tissue myofibroblasts and inquiescent and growing cultured fibroblasts. J Cell Biol 1993;1:103-111].

Myofibroblasts are essential for scar formation following myocardialinfarction (MI). However, their persistence can contribute to fibrosisand adverse myocardial remodeling, particularly if they remain active inotherwise healthy areas of the heart away from the site of injury. Thisreactive fibrosis is characterized by increased extracellular matrix andincreases the likelihood of arrhythmias [van den Borne S W, Diez J,Blankesteijn W M, Verjans J, Hofstra L, Narula J. Myocardial remodelingafter infarction: the role of myofibroblasts. Nat Rev Cardiol. 2010;7:30-7]. Similarly, the direct coupling of cardiomyocytes tomyofibroblasts increases the likelihood of arrhythmias, in contrast tonon-activated fibroblasts [Rohr S. Myofibroblasts in diseased hearts:new players in cardiac arrhythmias? Heart Rhythm. 2009; 6:848-56;Thompson S A, Copeland C R, Reich D H, Tung L. Mechanical couplingbetween myofibroblasts and cardiomyocytes slows electric conduction infibrotic cell monolayers. Circulation. 2011; 123:2083-93; Rosker C,Salvarani N, Schmutz S, Grand T, Rohr S. Abolishing myofibroblastarrhythmogeneicity by pharmacological ablation of alpha-smooth muscleactin containing stress fibers. Circulation Research. 2011;109:1120-31]. This reactive ongoing fibrosis leads to increasedmyocardial stiffness that contributes to systolic and diastolicdysfunction and heart failure progression [Pfeffer M A, Braunwald E.Ventricular remodeling after myocardial infarction. Experimentalobservations and clinical implications. Circulation. 1990; 81:1161-72;Swynghedauw B. Molecular mechanisms of myocardial remodeling. PhysiolRev. 1999; 79:215-62]. Over time, the density of myofibroblastsgenerally decreases following MI, however, these cells can persist insignificant numbers for years [Clanachan A S, Jaswal J S, Gandhi M,Bottorff D A, Coughlin J, Finegan B A, et al. Effects of inhibition ofmyocardial extracellular-responsive kinase and P38 mitogen-activatedprotein kinase on mechanical function of rat hearts after prolongedhypothermic ischemia. Transplantation. 2003; 75:173-80; Yada M,Shimamoto A, Hampton C R, Chong A J, Takayama H, Rothnie C L, et al.FR167653 diminishes infarct size in a murine model of myocardialischemia reperfusion injury. J Thorac Cardiovasc Surg. 2004; 128:588-94;Capano M, Crompton M. Bax translocates to mitochondria of heart cellsduring simulated ischaemia: involvement of AMP-activated and p38mitogen-activated protein kinases. Biochem J. 2006; 395:57-64; AleshinA, Sawa Y, Ono M, Funatsu T, Miyagawa S, Matsuda H. Myocardialprotective effect of FR167653; a novel cytokine inhibitor inischemic-reperfused rat heart. Eur J Cardiothorac Surg. 2004; 26:974-80;Gorog D A, Tanno M, Cao X, Bellahcene M, Bassi R, Kabir A M, et al.Inhibition of p38 MAPK activity fails to attenuate contractiledysfunction in a mouse model of low-flow ischemia. Cardiovascularresearch. 2004; 61:123-31].

After the initial myocardial cell death induced by ischemia, the heartquickly begins to promote the migration of fibroblasts. In the healingwound, fibroblasts proliferate and differentiate into myofibroblaststhat take on features resembling smooth muscle cells [Brown R D, AmblerS K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutic targetin myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 2005;45:657-87; Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiacfibroblast: therapeutic target in myocardial remodeling and failure.Annu Rev Pharmacol Toxicol. 2005; 45:657-87; Dobaczewski M,Gonzalez-Quesada C, Frangogiannis N G. The extracellular matrix as amodulator of the inflammatory and reparative response followingmyocardial infarction. J Mol Cell Cardiol. 2010; 48:504-11].

Extracellular Matrix Remodeling

Remodeling is broadly defined as changes in the organization of themyocardium, and is a critical process that allows the heart to adapt tochanges in mechanical, chemical and electrical signals [Brower G L,Chancey A L, Thanigaraj S, Matsubara B B, Janicki J S. Cause and effectrelationship between myocardial mast cell number and matrixmetalloproteinase activity. Am J Physiol Heart Circ Physiol. 2002; 283:H518-H525; Chancey A L, Brower G L, Janicki J S. Cardiac mastcell-mediated activation of gelatinase and alteration of ventriculardiastolic function. Am J Physiol Heart Circ Physiol. 2002; 282:H2152-H2158; Stewart J A Jr, Wei C C, Brower G L, Rynders P E, Hankes GH, Dillon A R, Lucchesi P A, Janicki J S, Dell'Italia L J. Cardiac mastcell- and chymase-mediated matrix metalloproteinase activity and leftventricular remodeling in mitral regurgitation in the dog. J Mol CellCardiol. 2003; 35: 311-319]. Cardiac fibroblasts are key components ofthis process, because of their ability to secrete and breakdown the ECM.Degradation of collagen requires the presence of matrixmetalloproteinases (MMPs) [Raffetto J D, Khalil R A. Matrixmetalloproteinases and their inhibitors in vascular remodeling andvascular disease. Biochem Pharmacol. 2008; 75: 346-359; Visse R, NagaseH. Matrix metalloproteinases and tissue inhibitors ofmetalloproteinases: structure, function, and biochemistry. Circ Res.2003; 92: 827-839]. In the normal heart, MMP expression and function aretightly regulated; however, in pathological states, MMP expression andactivity are increased, leading to excessive ECM degradation, which canhave profound effects on cardiac function. Following cardiac injury,fibroblast function can be influenced by chemical signals (e.g.,cytokines, matrikines and growth factors) in a paracrine or autocrinemanner. These factors can cause changes in fibroblast gene expression,as well as cell migration to the injured region to promote wound healingand scar formation.

Depending on the stage of heart failure, there can be considerablemyocyte hypertrophy and cell death. Dilatation can also be observed inlater stages; however, present at every stage are changes in the ECM,which are regulated by cardiac fibroblasts. There is also activation anddifferentiation of cardiac fibroblasts into myofibroblasts [Brown R D,Ambler S K, Mitchell M D, Long C S. The cardiac fibroblast: therapeutictarget in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol.2005; 45: 657-687; Weber K T. Fibrosis in hypertensive heart disease:focus on cardiac fibroblasts. J Hypertens. 2004; 22: 47-50;Frangogiannis N G, Michael L H, Entman M L. Myofibroblasts inre-perfused myocardial infarcts express the embryonic form of smoothmuscle myosin heavy chain. Cardiovasc Res. 2000; 48: 89-100]. Aftermaturation to myofibroblasts, an increase in the synthesis and secretionof fibronectin is observed [Gabbiani G. The myofibroblast in woundhealing and fibrocontractive diseases. J Pathol. 2003; 200: 500-503]. Asthe heart undergoes remodeling associated with heart failure, anincrease in cytokine and growth factor secretion is observed. Inresponse to these various factors, myofibroblasts begin to proliferate,migrate and remodel the cardiac interstitium through increased secretionof MMPs and collagen [Brown R D, Ambler S K, Mitchell M D, Long C S. Thecardiac fibroblast: therapeutic target in myocardial remodeling andfailure. Annu Rev Pharmacol Toxicol. 2005; 45: 657-687; Weber K T.Fibrosis in hypertensive heart disease: focus on cardiac fibroblasts. JHypertens. 2004; 22: 47-50; Lindsey M L, Escobar G P, Mukherjee R,Goshorn D K, Sheats N J, Bruce J A, Mains I M, Hendrick J K, Hewett K W,Gourdie R G, Matrisian L M, Spinale F G. Matrix metalloproteinase-7affects connexin-43 levels, electrical conduction, and survival aftermyocardial infarction. Circulation. 2006; 113: 2919-2928; Raizman J E,Komijenovic J, Chang R, Deng C, Bedosky K M, Rattan S G, Cunnington R H,Freed D H, Dixon I M. The participation of the Na+-Ca2+ exchanger inprimary cardiac myofibroblast migration, contraction and proliferation.J Cell Physiol. 2007; 213: 540-551]. To further stimulate the remodelingprocess, cardiac fibroblasts secrete increased amounts of growth factorsand cytokines, specifically IL-1β, IL-6, and tumor necrosis factor(TNF)-α, which, in turn, activate MMPs, leading to further cardiacremodeling [Brown R D, Ambler S K, Mitchell M D, Long C S. The cardiacfibroblast: therapeutic target in myocardial remodeling and failure.Annu Rev Pharmacol Toxicol. 2005; 45: 657-687; Corda S, Samuel J L,Rappaport L. Extracellular matrix and growth factors during heartgrowth. Heart Fail Rev. 2000; 5: 119-130; Brown R D, Mitchell M D, LongC S. Proinflammatory cytokines and cardiac extracellular matrix:regulation of fibroblast phenotype. In: Villarreal F J, ed. InterstitialFibrosis in Heart Disease. New York: Springer; 2004: 57-81]. Initially,all of these changes are critical to the reparative wound healingresponse. However, over time, these changes become maladaptive leadingto fibrosis and reduced cardiac function.

Although not present in normal myocardium, myofibroblasts are highlylocalized to sites of injury where synthesis and deposition of collagenpromotes scar formation and fibrosis [Sun Y, Weber K T. Infarct scar: adynamic tissue. Cardiovasc Res. 2000; 46: 250-256]. In addition, thesecells are also located near, or associated with, blood vessels. Becausemyofibroblasts express contractile proteins, such as smooth muscleactin, they are able to provide mechanical tension to the remodelingmatrix, helping to close the wound and reduce scarring [Gabbiani G. Thecellular derivation and the life span of the myofibroblast. Pathol ResPract. 1996; 192: 708-711; Brown E, Dejana E. Cell-to-cell contract andthe extracellular matrix. Curr Opin Cell Biol. 2003; 15: 505-508;Gabbiani G. The myofibroblast in wound healing and fibrocontractivediseases. J Pathol. 2003; 200: 500-503]. As the scar matures, cells inthe scar undergo apoptosis, leaving a scar that consists mainly ofcollagen and ECM proteins, but myofibroblasts are still present [GurtnerG C, Werner S, Barrandon Y, Longaker M T. Wound repair and regeneration.Nature. 2008; 453: 314-321]. Myofibroblasts have been observed in maturescars in a rat model of myocardial infarct, as well as in scarred humantissue [Sun Y, Weber K T. Infarct scar: a dynamic tissue. CardiovascRes. 2000; 46: 250-256; Willems I E, Havenith M G, DeMey J G, Daemen MJ. The alpha-smooth muscle actin-positive cells in healing humanmyocardial scars. Am J Pathol. 1994; 145: 868-875]. It is not known whymyofibroblasts persist, but they are highly involved in regulatingcardiac remodeling, cardiac dysfunction, and ultimately cardiac failure.

In the normal heart, collagen and other ECM components help maintainheart structure and function. ECM is synthesized and degraded by cardiacfibroblasts in a coordinated fashion; however, during heart failurethere is disruption of these regulatory pathways, leading to animbalance of ECM synthesis and degradation that determines the level ofcardiac remodeling. Increases in the extracellular matrix or fibrosismay be reparative, replacing areas of myocyte loss with a structuralscar, or reactive, involving increases in ECM deposition at sites otherthan those of the primary injury. Fibroblast proliferation anddifferentiation to myofibroblasts in remote areas of the infarct(reactive fibrosis) can cause an increase in ECM synthesis anddeposition which results in increased mechanical stiffness andcontributes to relaxation abnormalities, arrhythmogenicity, progressivediastolic dysfunction and heart failure. The size of the infarcted area,the infarcted wound healing, and chronic left ventricular (LV)remodeling determine the extent of heart failure that results [Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction.Experimental observations and clinical implications. Circulation. 1990;81:1161-72; Opie L H, Commerford P J, Gersh B J, Pfeffer M A.Controversies in ventricular remodelling. Lancet. 2006; 367:356-67; DornG W, 2nd. Novel pharmacotherapies to abrogate postinfarction ventricularremodeling. Nat Rev Cardiol. 2009; 6:283-91]. Progressive increases infibrosis can lead to systolic dysfunction and left ventricularhypertrophy. Moreover, increased levels of collagen can disruptelectrophysiological communication between myocytes. Furthermore,perivascular fibrosis can impair myocyte oxygen supply, reduce coronaryreserve, and accentuate ischemia [Brown R D, Ambler S K, Mitchell M D,Long C S. The cardiac fibroblast: therapeutic target in myocardialremodeling and failure. Annu Rev Pharmacol Toxicol. 2005; 45:657-87].

Anti-fibrosis strategies are limited and are not particularly targeted.Currently, angiotensin-converting enzyme (ACE) inhibition, angiotensinreceptor antagonism, and HMG-CoA-reductase inhibition are available[Opie L H, Commerford P J, Gersh B J, Pfeffer M A. Controversies inventricular remodeling. Lancet. 2006; 367:356-67; Bauersachs J, GaluppoP, Fraccarollo D, Christ M, Ertl G. Improvement of left ventricularremodeling and function by hydroxymethylglutaryl coenzyme a reductaseinhibition with cerivastatin in rats with heart failure after myocardialinfarction. Circulation. 2001; 104:982-5; Shyu K G, Wang B W, Chen W J,Kuan P, Hung C R. Mechanism of the inhibitory effect of atorvastatin onendoglin expression induced by transforming growth factorbetalincultured cardiac fibroblasts. Eur J Heart Fail. 2010; 12:219-26]. Whilethese have shown some beneficial effects, more effective preventionfocused at the level of the fibroblast is needed [Brown R D, Ambler S K,Mitchell M D, Long C S. The cardiac fibroblast: therapeutic target inmyocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 2005;45:657-87; Fraccarollo D, Galuppo P, Bauersachs J. Novel therapeuticapproaches to post-infarction remodelling. Cardiovascular research.2012; 94:293-303]. The Role of the TGFβ/p38 MAPK-MK2 Signaling Pathwayin Fibrosis and Post-MI Remodeling

The TGFβ/p38 pathway is central to the pathogenesis of fibrosis. MAPKAPkinase 2 (MK2) is a downstream signaling molecule in the TGFβ/p38pathway and MK2 phosphorylates and activates signaling molecules thatare important in the pathologic processes of fibrotic disease, includinginflammatory signaling and fibroblast activation and migration (FIG.13). MK2 is upstream of both fibrosis and inflammatory pathways. Thefibrosis pathway leads to increases in stress fibers (α-smooth muscleactin expression) which results in the myofibroblast phenotype.Myofibroblasts accumulate at sites of tissue remodeling and produceextracellular matrix components such as collagen and hyaluronan (HA)that ultimately compromise organ function. MK2 also phosphorylatestranscription factors such as hnRNPA0 which stabilizes cytokines in theinflammatory pathway.

p38 mitogen-activated protein kinase (MAPK) and its upstream anddownstream signaling molecules have been shown to play an important rolein the response to cellular stress from stimuli [Saklatvala, Curr OpinPharmacol, 4:372-377, 2004; Edmunds, J. and Talanian, MAPKAP Kinase 2(MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J andLaufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the RoyalSociety of Chemistry, 2012].

There are four isoforms of p38 (i.e., p38α, p38β, p38γ, and p38δ) withp38α being most clearly associated with inflammation. Cytokines andother extracellular stimuli (such as growth factors, DNA damage, andoxidative stress) signal through multiple receptors and other mechanismsto activate a cascade of kinases starting with a MAP3K (e.g., MEKK3 orTAK1), then a MAP2K (e.g., MKK3 or MKK6), and then a MAPK (such asp38α). By direct and indirect effects, including the stabilization,translocation, and translation of mRNAs, p38 plays a maj or role in theproduction of pro-inflammatory cytokines, such as TNF-α, IL-6, andIFN-γ, as well as the induction of other pro-inflammatory cytokines,such as COX-2.

Generally, in resting cells, p38 MAPK and MK2 are physically boundtogether in the nucleus. Cellular stress causes the phosphorylation ofp38 MAPK by an upstream kinase, such as MKK3 [Kim et al., Am J PhysiolRenal Physiol, 292: F1471-1478, 2007]. The activated p38 MAPK thenphosphorylates MK2 at residues Thr-222, Ser-272, and/or Thr-334 [Engelet al., EMBO J, 17: 3363-3371, 1998]. The activated MK2 and p38, stillphysically bound together, translocate to cytoplasm, where theyphosphorylate their respective target protein [Ben-Levy et al., CurrBiol, 8:1049-1057, 1998].

In turn, activated MK2 mediates phosphorylation of HSPB1 in response tostress, leading to dissociation of HSPB1 from large small heat-shockprotein (sHsps) oligomers, thereby impairing their chaperone activitiesand ability to protect against oxidative stress effectively. MK2 is alsoinvolved in inflammatory and immune responses by regulating TumorNecrosis Factor (TNF) and IL-6 production post-transcriptionally. Thisactivity is mediated by phosphorylation of Adenine- and Uridine(AU)-Rich Elements (AREs)-binding proteins, such as Embryonic Lethal,Abnormal Vision, Drosophila-Like 1 (ELAVL1), Heterogeneous NuclearRibonucleoprotein AO (HNRNPA0), Polyadenylate-Binding Protein 1(PABPC1), and Tristetraprolin (TTP/ZFP36), which, in turn, regulate thestability and translation of TNF-α and IL-6 mRNAs. Phosphorylation ofTTP/ZFP36, a major post-transcriptional regulator of TNF-α, promotes itsbinding to 14-3-3 proteins and reduces its affinity to ARE mRNA, therebyinhibits degradation of ARE-containing transcript.

In addition, MK2 also plays an important role in the late G2/Mcheckpoint following DNA damage through a process ofpost-transcriptional mRNA stabilization. Following DNA damage, MK2re-localizes from nucleus to cytoplasm and phosphorylates HeterogeneousNuclear Ribonucleoprotein AO (HNRNPA0) and Poly(A)-specific Ribonuclease(PARN), leading to stabilization of growth arrest andDNA-damage-inducible protein 45A (GADD45A) mRNA. Additionally, studieshave shown that MK2 is involved in the toll-like receptor signalingpathway (TLR) in dendritic cells and in acute TLR-inducedmacropinocytosis by phosphorylating and activating Ribosomal protein S6kinase, 90 kDa, polypeptide 3 (RPS6KA3).

Although enzymes at each level of the aforementioned p38 MAPK signalingcascade have been explored for anti-cytokine drug discovery, it isdifficult to generalize how upstream or downstream targets in such apathway might vary in their potential for efficacy. For example,upstream targets might have multiple effects, enhancing efficacy, butmight be bypassed by other signaling mechanisms, limiting the impact ofinhibition. Undesirable side-effects are similarly difficult to predict.Therefore, specific properties of signaling mechanisms like that of thep38 pathway must be considered case by case to select the best targetsbased on empirical experience [Edmunds, J. and Talanian, MAPKAP Kinase 2(MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J andLaufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the RoyalSociety of Chemistry, 2012].

Indeed, while there have been many reports of p38 inhibitors withpromising properties in vitro and in animal models of disease, none haveachieved clinical success [Edmunds, J. and Talanian, MAPKAP Kinase 2(MK2) as a Target for Anti-inflammatory Drug Discovery. In Levin, J andLaufer, S (Ed.), RSC Drug Discovery Series No. 26, p 158-175, the RoyalSociety of Chemistry, 2012]. Many targets beyond those related tocytokine production are regulated by p38, consistent with observedpleiotropic consequences of its inhibition and suggesting multiplemechanisms of toxicity and even pro-inflammatory effects. For example,in hepatocytes, p38 directly and indirectly down-regulates JNK, therebymodulating hepatocyte sensitivity to lipopolysaccharide (LPS) and TNF-αinduced cell death; this may be an important mechanism of p38inhibition-induced liver toxicity. In addition, activation of MSK1 andMSK2 by p38 may induce expression of anti-inflammatory cytokine IL-10,and therefore inhibition of p38 may have a pro-inflammatory effect thatcontributes to the observed transient suppression of inflammatorymarkers by p38 inhibitors. Thus, there are significant concerns that, asan anti-inflammatory strategy, p38 inhibition will not result inadequate efficacy or acceptable safety.

On the other hand, MK2 attracted wide attention as a potential drugdiscovery target when it was reported that MK2-deficient knockout miceare viable and fertile, and are defective in TNF-α production.Splenocytes derived from these animals are defective in the productionof several pro-inflammatory cytokines, including TNF-α, IL-6 and IFN-γand the animals themselves are resistant to collagen-induced arthritis(a mouse model of rheumatoid arthritis (RA)), as well as inovalbumin-induced airway inflammation (a mouse model of asthma). Dosedorally, inhibitors of MK2 can block acute systemic induction of TNF-α byLPS in rats and can reduce paw swelling in the rat streptococcal cellwall (SCW)-induced arthritis model. These results suggested that MK2mediates most or all inflammatory signals of the p38 cascade while otherp38 substrates regulate the pathways responsible for toxicity orattenuated efficacy; and that MK2 inhibition might deliver on thepromise of p38 inhibition for anti-inflammatory efficacy while alsogiving a more favorable safety profile.

Myocyte death during lethal myocardial infarction, cardiac dysfunction,and fibrosis during post-MI remodeling and hypertrophy is associatedwith sustained activation of p38 [Clark J E, Sarafraz N, Marber M S.Potential of p38-MAPK inhibitors in the treatment of ischaemic heartdisease. Pharmacol Ther. 2007; 116:192-206; Kerkela R, Force T. p38mitogen-activated protein kinase: a future target for heart failuretherapy? Journal of the American College of Cardiology. 2006; 48:556-8;Wang Y. Mitogen-activated protein kinases in heart development anddiseases. Circulation. 2007; 116:1413-23]. Recent studies in MK2−/− micehave illustrated that MK2 acts downstream of p38 and is responsible forp38-induced heart failure [Streicher J M. The role of mitogen activatedprotein kinase activated protein kinase-2 in regulating p38 mitogenactivated protein kinase induced cyclooxygenase-2 induction and heartfailure: University of California-Los Angeles; 2009]. Similarly, MK2−/−mice are resistant to ischemia reperfusion injury [Shiroto K, Otani H,Yamamoto F, Huang C K, Maulik N, Das D K. MK2−/− gene knockout mousehearts carry anti-apoptotic signal and are resistant to ischemiareperfusion injury. J Mol Cell Cardiol. 2005; 38:93-7], indicating acritical role of MK2 in ischemic heart disease experimentally. When micelacking MK2 (MK2−/−) were compared to M2+/+ mice on a transgenic p38background, the transgenic p38-induced heart failure in the MK2−/− micewas significantly protective [Streicher J M. The role of mitogenactivated protein kinase activated protein kinase-2 in regulating p38mitogen activated protein kinase induced cyclooxygenase-2 induction andheart failure: University of California-Los Angeles; 2009]. Similarly,MK2−/− mice are resistant to ischemia reperfusion injury [Shiroto K,Otani H, Yamamoto F, Huang C K, Maulik N, Das D K. MK2−/− gene knockoutmouse hearts carry anti-apoptotic signal and are resistant to ischemiareperfusion injury. J Mol Cell Cardiol. 2005; 38:93-7], implicating acritical role of MK2 in ischemic injury. Consistent with an MK2-p38 axismediating ischemic cardiac damage, inhibiting p38 activation protectsthe heart against ischemic insult and cardiac dysfunction [Marber M S,Rose B, Wang Y. The p38 mitogen-activated protein kinase pathway—apotential target for intervention in infarction, hypertrophy, and heartfailure. J Mol Cell Cardiol. 2011; 51:485-90; Tanno M, Bassi R, Gorog DA, Saurin A T, Jiang J, Heads R J, et al. Diverse mechanisms ofmyocardial p38 mitogen-activated protein kinase activation: evidence forMKK-independent activation by a TAB 1-associated mechanism contributingto injury during myocardial ischemia. Circulation Res. 2003; 93:254-61;Marais E, Genade S, Huisamen B, Strijdom J G, Moolman J A, Lochner A.Activation of p38 MAPK induced by a multi-cycle ischaemicpreconditioning protocol is associated with attenuated p38 MAPK activityduring sustained ischemia and reperfusion. J Mol Cell Cardiol. 2001;33:769-78; Sanada S, Kitakaze M, Papst P J, Hatanaka K, Asanuma H, AkiT, et al. Role of phasic dynamism of p38 mitogen-activated proteinkinase activation in ischemic preconditioning of the canine heart.Circulation Res. 2001; 88:175-80; Nagarkatti D S, Sha'afi RI. Role ofp38 MAP kinase in myocardial stress. J Mol Cell Cardiol. 1998;30:1651-64]. At the cellular level, ischemic activation of the MK2-p38signalling pathway induces cardiac apoptosis [Matsumoto-Ida M, TakimotoY, Aoyama T, Akao M, Takeda T, Kita T. Activation of TGF-beta1-TAK1-p38MAPK pathway in spared cardiomyocytes is involved in left ventricularremodeling after myocardial infarction in rats. Am J Physiol HeartCircul Physiol. 2006; 290:H709-15], specifically in cardiomyocytes[Clark J E, Sarafraz N, Marber M S. Potential of p38-MAPK inhibitors inthe treatment of ischaemic heart disease. Pharmacol Ther. 2007;116:192-206; Kerkela R, Force T. p38 mitogen-activated protein kinase: afuture target for heart failure therapy? Journal of the American Collegeof Cardiology. 2006; 48:556-8; Wang Y. Mitogen-activated protein kinasesin heart development and diseases. Circulation. 2007; 116:1413-23]. Infibroblasts, p38 regulates extracellular matrix proteins in primarycardiac fibroblasts during oxidative stress [Hsu P L, Su B C, Kuok Q Y,Mo F E. Extracellular matrix protein CCN1 regulates cardiomyocyteapoptosis in mice with stress-induced cardiac injury. CardiovascularRes. 2013; 98:64-72].

The use of rationally designed cell-permeable peptides that inhibitMitogen Activated Protein Kinase Activated Protein Kinase II (MK2)activity and downstream fibrosis and inflammation is unique. Recentstudies have reported that the cell-permeable peptide MMI-0100 inhibitsinflammation and fibrosis in intimal hyperplasia in a mouse vein graftmodel [Muto A, Panitch A, Kim N, Park K, Komalavilas P, Brophy C M, etal. Inhibition of Mitogen Activated Protein Kinase Activated ProteinKinase II with MMI-0100 reduces intimal hyperplasia ex vivo and in vivo.Vascular Pharmacol. 2012; 56:47-55], bleomycin-induced pulmonaryfibrosis [Vittal R, Fisher A, Gu H, Mickler E A, Panitch A, Lander C, etal. Peptide-mediated Inhibition of MK2 Ameliorates Bleomycin-InducedPulmonary Fibrosis. Am J Respir Cell Mol Biol. 2013] and in inhibitingabdominal adhesions post-surgery [Ward B C, Kavalukas S, Brugnano J,Barbul A, Panitch A. Peptide inhibitors of MK2 show promise forinhibition of abdominal adhesions. J Surg Res 2011; 169:e27-36]. Thesepeptides target the substrate-binding site of MK2 and contain permeantdomains that are rapidly taken up by macropinocytosis and targeted toendosomal compartments, where they are retained for up to 7 days [FlynnC R, Cheung-Flynn J, Smoke C C, Lowry D, Roberson R, Sheller M R, et al.Internalization and intracellular trafficking of a PTD-conjugatedanti-fibrotic peptide, AZX100, in human dermal keloid fibroblasts. JPharm Sci. 2010; 99:3100-21].

To minimize the extent of heart failure after a large or recurrent MI,therapeutic strategies are needed to limit infarct wound healing. Thedescribed invention offers approaches to minimize the extent of heartfailure or recurrent MI by utilizing a cell-penetrating, peptide-basedinhibitor of MK2.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides a method forreducing left ventricular remodeling resulting from a myocardialinfarction (MI) in a subject in need of such treatment, the methodcomprising administering to the subject a therapeutic amount of apharmaceutical composition comprising a polypeptide of amino sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent thereofmade from a fusion between a first polypeptide that is a cell permeableprotein (CPP) selected from the group consisting of a polypeptide ofamino acid sequence YARAAARQARA (SEQ ID NO: 2), WLRRIKAWLRRIKA (SEQ IDNO: 21), WLRRIKA (SEQ ID NO: 22), YGRKKRRQRRR (SEQ ID NO: 23),FAKLAARLYR (SEQ ID NO: 25), and KAFAKLAARLYR (SEQ ID NO: 26), and asecond polypeptide that is a therapeutic domain (TD), and apharmaceutically acceptable carrier, the ventricular remodeling beingcharacterized by aberrant deposition of an extracellular matrix protein,an aberrant promotion of fibroblast proliferation in the heart, anaberrant induction of myofibroblast differentiation, an aberrantpromotion of attachment of myofibroblasts to an extracellular matrix ora combination thereof; wherein the therapeutic amount is effective (i)to inhibit MK2; (ii) to reduce cardiac fibrosis; (iii) to preservecardiac muscle; and (iv) to preserve systolic function, wherein themethod is effective to protect cardiomyocytes from an ischemic insult,reduce regions of fibrosis at a site of ischemic insult, or acombination thereof, wherein without the therapeutic amount, theventricular remodeling can progress to heart failure.

According to another aspect, the described invention provides a methodfor preserving cardiac function after a myocardial infarction (MI) in asubject in need thereof, the method comprising administering to thesubject in need thereof a therapeutic amount of a pharmaceuticalcomposition comprising a polypeptide of amino sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent thereofmade from a fusion between a first polypeptide that is a cell permeableprotein (CPP) selected from the group consisting of a polypeptide ofamino acid sequence YARAAARQARA (SEQ ID NO: 2), WLRRIKAWLRRIKA (SEQ IDNO: 21), WLRRIKA (SEQ ID NO: 22), YGRKKRRQRRR (SEQ ID NO: 23),FAKLAARLYR (SEQ ID NO: 25), and KAFAKLAARLYR (SEQ ID NO: 26), and asecond polypeptide that is a therapeutic domain (TD), and apharmaceutically acceptable carrier, wherein the therapeutic amount iseffective (i) to inhibit MK2; (ii) to increase ejection fraction; (iii)to increase fractional shortening; and (iv) to decrease left ventriculardilation, wherein the method is effective to protect cardiomyocytes froman ischemic insult, reduce regions of fibrosis at a site of ischemicinsult, or a combination thereof.

According to one embodiment, the therapeutic domain (TD) is selectedfrom the group consisting of a polypeptide of amino acide sequenceKALARQLGVAA (SEQ ID NO: 3), KALARQLAVA (SEQ ID NO: 9), KALARQLGVA (SEQID NO: 10), KALARQLGVAA (SEQ ID NO: 11), KALNRQLGVAA (SEQ ID NO: 12),KAANRQLGVAA (SEQ ID NO: 13), KALNAQLGVAA (SEQ ID NO: 14), KALNRALGVAA(SEQ ID NO: 15), KALNRQAGVAA (SEQ ID NO: 16), KALNRQLAVA (SEQ ID NO:17), KALNRQLAVAA (SEQ ID NO: 18), KALNRQLGAAA (SEQ ID NO: 19), andKALNRQLGVA (SEQ ID NO: 20).

According to one embodiment, the cardiac fibrosis is reduced by 50%compared to an untreated control subject.

According to one embodiment the therapeutic amount is effective toinhibit apoptotic cell death of cardiomyocytes in a peri-infarct zone.According to one such embodiment, the therapeutic amount is effective toinhibit apoptotic cell death of cardiomyocytes, enhance apoptotic celldeath of cardiac fibroblasts, or a combination thereof.

According to one embodiment, the therapeutic amount is effective toinhibit caspase activity. According to another embodiment, the caspaseactivity is caspase 3/7 activity.

According to one embodiment, the therapeutic amount is effective toenhance lactate dehydrogenase (LDH) release.

According to one embodiment, the therapeutic amount is effective toinhibit heterogeneous nuclear ribonucleoprotein AO (HNRNPA0) proteinexpression.

According to another aspect, the described invention provides a kitcomprising: (a) a composition comprising at least one MK2 inhibitorpeptide; (b) a means for administering the composition; and (c) apackaging material.

According to one embodiment, the MK2 inhibitor peptide is a polypeptideof amino sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to one embodiment, the composition further comprises apharmaceutically acceptable excipient.

According to one embodiment, the packaging material is an instruction.

According to one embodiment, the means for administering the compositioncomprises a syringe. According to another embodiment, the means foradministering the composition comprises an inhalation device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show an experimental design for treatment of acute myocardialinfarction (AMI) with 50 μg/kg/day MMI-0100 peptide or PBS administeredintra-peritoneally 30 minutes after insults with survival analysis. FIG.1A. Echocardiography schedule in relation to acute myocardial infarction(AMI) and thoracotomy and sham ligation surgical intervention.

FIG. 1B. Daily drug delivery schedule of MMI-0100 peptide or PBScontrol. FIG. 1C. Conscious echocardiographic analysis of AMI(thoracotomy and permanent LAD coronary artery ligation), thoracotomyand sham ligation (threaded, but not tied, removed), and AMI followed byMMI-0100 peptide administered intra-peritoneally in PBS vehicle, started30 minutes after permanent LAD ligation placed. FIG. 1D. RepresentativeM-mode tracing from three experimental groups at baseline, 1 week, and 2weeks after AMI surgical intervention. A Kruskal-Wallis Oneway ANOVA wasperformed at each terminal time point in experiments performed inparallel. If significance was reached (p<0.05), a post-hoc all pairwiseMultiple Comparison Procedures (Tukey Test) was performed between eachof the groups to determine significance. *p<0.05 vs. thoracotomy andsham ligation (control); **p<0.05 vs. other two groups.

FIGS. 2A-E show permanent LAD coronary artery ligation (surgicalinduction of acute MI) treated daily with 50 μg/kg/day MMI-0100 peptide(first given 30 minutes post-AMI) in vivo results in a significantreduction in fibrosis at day 14. FIG. 2A. The area of fibrosis wasanalyzed in 3-4 blindly chosen hearts each heart at 14-15 levels, 3sections at each level, and blinded fibrosis analysis of trichromestained histological sections using Aperio (42 sections analyzed permouse heart). FIG. 2B. Histological analysis of fibrosis (collagenstaining blue by Aperio algorithm analysis) of 3-4 hearts per groupresulting from acute myocardial infarction at 14 days post-AMI. FIG. 2C.Representative trichrome stained sections from mouse hearts challengedwith permanent AMI (˜21% of the area stains blue, including primarilyinterstitial fibrosis). FIG. 2D. Representative trichrome-stainedsections from mouse hearts challenged with permanent AMI treated dailywith 50 μg/kg/day MMI-0100 peptide starting 30 minutes post-infarction(˜11% of the area stains blue, including primarily interstitialfibrosis). FIG. E. Representative trichrome-stained sections from mousecontrol hearts, including the thoracotomy+sham ligation, no surgery+nodrug, and no surgery+50 μg/kg/day MMI-0100 peptide. (0.9% of the areastains blue, representing connective tissue and vessels; no interstitialfibrosis evident in any analyzed section). A Kruskal-Wallis One-wayANOVA was performed on the fibrosis % from serial sections using 3-4hearts per group; each single fibrosis per heart was the weighted meanof 126-180 sections described in FIG. 2A. above. If significance wasreached (p<0.05), a post-hoc all pairwise Multiple Comparison Procedures(Tukey Test) was performed between each of the groups to determinesignificance. *p<0.05 vs. all other groups.

FIGS. 3A-C show that MMI-0100 peptide reduces caspase 3/7 activation inH9C2 cardiomyocytes challenged with 1% hypoxia. FIG. 3A. H9C2 cells werechallenged with 1% hypoxia for 8, 16, and 24 hours; the media werecollected for LDH release and cells immediately harvested for caspase3/7 activity and Western blot analysis at each time point. Each barrepresents 3 wells performed in triplicate in experimental conditionsrepeated on at least 2 independent occasions. FIG. 3B. Caspase 3/7activity of harvested cells at 8, 16, and 24 hours in the presence orabsence of MMI-0100 peptide; 0 μM (vehicle only, 0.1% DMSO final), 20μM, or 100 μM MMI-0100 peptide. FIG. 3C. LDH detection in media from thesame experimental conditions as the caspase activity described above. AKruskal-Wallis Oneway ANOVA was performed at each terminal time point inexperiments run in parallel. If significance was reached (p<0.05), apost-hoc all pairwise Multiple Comparison Procedures (Tukey Test) wasperformed between each of the groups to determine significance. *p<0.05vs. 0 μM group (DMSO vehicle control); **p<0.05 vs. other two groups.

FIGS. 4A-B show that MMI-0100 peptide reduces MK2 activity in H9C2cardiomyocytes challenged with 1% hypoxia as measured by downstreamHNRNPA0 protein expressed, but does not reduce phospho- or total MK2levels. FIG. 4A. H9C2 cells were challenged with 1% hypoxia for 8, 16,and 24 hours; desitometric analysis of Western immunoblot (right,representative 1 of 3 replicates per bar) demonstrated significantdecreases in HNRNPA0 protein expression. FIG. 4B. Densitometric analysisphospho- and total MK2 levels (below). A Kruskal-Wallis One-way ANOVAwas performed at each terminal time point in experiments run inparallel. If significance was reached (p<0.05), a post-hoc all pairwiseMultiple Comparison Procedures (Tukey Test) was performed between eachof the groups to determine significance. **p<0.05 vs. other 2 groups.

FIGS. 5A-C show that MMI-0100 peptide reduces caspase 3/7 activation inHL1 cardiomyocytes challenged with 1% hypoxia. FIG. 5A. HL1 cells werechallenged with 1% hypoxia for 4, 8, and 12 hours; the media werecollected for LDH release and cells immediately harvested for caspase3/7 activity and Western blot analysis at each time point. Each barrepresents 3 wells performed in triplicate in experimental conditionsrepeated on at least 2 independent occasions. FIG. 5B. Caspase 3/7activity of harvested cells at 4, 8, and 12 hours in the presence orabsence of MMI-0100 peptide; 0 LM (vehicle only, 0.1% DMSO final), 20 Mand 100 LM MMI-0100 peptide. FIG. 5C. LDH detection in media from thesame experimental conditions as the caspase activity described above. AKruskal-Wallis Oneway ANOVA was performed at each terminal time point inexperiments run in parallel. If significance was reached (p<0.05), apost-hoc all pairwise Multiple Comparison Procedures (Tukey Test) wasperformed between each of the groups to determine significance. *p<0.05vs. 0 LM group (DMSO vehicle control); **p<0.05 vs. other two groups;*** p<0.05 vs. 20 μM MMI-0100 peptide group.

FIGS. 6A-B show that MMI-0100 peptide reduces MK2 activity in HL1cardiomyocytes challenged with 1% hypoxia as measured by downstreamHNRNPA0 protein expressed, but does not reduce phospho- or total MK2levels. FIG. 6A. H9C2 cells were challenged with 1% hypoxia for 4, 8,and 12 hours; desitometric analysis of Western immunoblot (right,representative 1 of 3 replicates per bar) demonstrated significantdecreases in HNRNPA0 protein expression. FIG. 6B. Densitometric analysisphospho- and total MK2 levels (below). A Kruskal-Wallis One-way ANOVAwas performed at each terminal time point in experiments run inparallel. If significance was reached (p<0.05), a post-hoc all pairwiseMultiple Comparison Procedures (Tukey Test) was performed between eachof the groups to determine significance. *p<0.05 vs. 0 LM group (DMSOvehicle control).

FIGS. 7A-C show that MMI-0100 peptide enhances caspase 3/7 activationand LDH release in primary cardiac fibrolasts challenged with 1%hypoxia. FIG. 7A. Primary cardiac fibroblasts were challenged with 1%hypoxia for 16, 32, and 48 hours; the media was collected for LDHrelease and cells immediately harvested for caspase 3/7 activity andWestern blot analysis at each time point. Each bar represents 3 wellsperformed in triplicate in experimental conditions repeated on at least2 independent occasions. FIG. 7B. Caspase 3/7 activity of harvestedcells at 16, 32, and 48 hours in the presence or absence of MMI-0100peptide; 0 LM (vehicle only, 0.1% DMSO final), 20 μM, or 100 μM MMI-0100peptide. FIG. 7C. LDH detection in media from the same experimentalconditions as the caspase activity described above. A Kruskal-WallisOne-way ANOVA was performed at each terminal time point in experimentsrun in parallel. If significance was reached (p<0.05), a post-hoc allpairwise Multiple Comparison Procedures (Tukey Test) was performedbetween each of the groups to determine significance. *p<0.05 vs. 0 μMgroup (DMSO vehicle control); **p<0.05 vs. other two groups; *** p<0.05vs. 20 μM MMI-0100 peptide group.

FIGS. 8A-B show that MMI-0100 peptide reduction of MK2 activity is notdetected at the time points cell death is affected in primary cardiacfibroblasts challenged with 1% hypoxia, as measured by downstreamHNRNPA0, phospho-MK2, and total MK2 protein levels. FIG. 8A. Primarycardiac fibroblasts were challenged with 1% hypoxia for 16, 32, and 48hours; desitometric analysis of Western immunoblot (right,representative 1 of 3 replicates per bar) no significant changes inHNRNPA0 protein expression were detected. FIG. 8B. Densitometricanalysis phospho- and total MK2 levels (below). A Kruskal-Wallis OnewayANOVA was performed at each terminal time point in experiments run inparallel. n.s.=not significant.

FIGS. 9A-B show survival analysis after permanent LAD coronary arteryligation and after 50 μg/kg/day MMI-0100 peptide in PBS intraperitonealinjection. FIG. 9A. Kaplan-Meier survival curve post-surgery of acute MIand acute MI+MMI-0100 peptide. FIG. 9B. Kaplan-Meier survival curveafter daily MMI-0100 peptide or PBS (vehicle only) treatment. A Log-rank(Mantel-Cox) test was performed to test differences in survival (Chisquare 0.9211, df=1, p=0.3372).

FIGS. 10A-F show histological analysis of hearts from mice challengedwith AMI and treated with PBS or 50 μg/kg/day MMI-0100 peptide (PBSvehicle) which reveals sparing of cells within the fibrotic scarring ofthe anterior wall. FIG. 10A-C: Representative sections reveal few cellsremain in the fibrotic scar of the anterior wall of AMI hearts. FIG.10D-F: Representative sections reveal variable periodic islands of cellsin the anterior wall scar. Magnification 4-8×, with scale on eachindividual panel.

FIGS. 11A-C show that cardiomyocyte cell lines and primary cardiacfibroblasts treated with 20 or 100 mM MMI-0100 peptide have an enhancedLDH release during hypoxia challenge, compared to their response duringnormoxia (FIG. 3C, FIG. 5C, FIG. 7C). H9C2 cells (FIG. 11A.); HL1 cells(FIG. 11B.); and primary cardiac fibroblasts (FIG. 11C.) do not exhibitan enhanced LDH release at the primary time point tested with hypoxia(8, 4, and 16 hours, respectively). Significant increases in LDH aredetected (compare to FIG. 3C, FIG. 5C, and FIG. 7C, respectively). AKruskal-Wallis One-way ANOVA was performed at each terminal time pointin experiments run in parallel. If significance was reached (p<0.05), apost-hoc all pairwise Multiple Comparison Procedures (Tukey Test) wasperformed between each of the groups to determine significance. *p<0.05vs. 0 μM group (DMSO vehicle control).

FIG. 12 shows a schematic of the p38 MAPK signaling pathway (taken frommoeraematrix.com).

FIG. 13 shows a schematic of the Mapkap kinase 2 (MK2) pathway.

DETAILED DESCRIPTION OF THE INVENTION

The described invention can be better understood from the followingdescription of exemplary embodiments, taken in conjunction with theaccompanying figures and drawings. It should be apparent to thoseskilled in the art that the described embodiments provided herein aremerely exemplary and illustrative and not limiting.

Definitions

Various terms used throughout this specification shall have thedefinitions set out herein.

The term “administering” as used herein includes in vivo administration,as well as administration directly to tissue ex vivo. Generally,compositions can be administered systemically either orally, buccally,parenterally, topically, by inhalation or insufflation (i.e., throughthe mouth or through the nose), or rectally in dosage unit formulationscontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired, or can be locally administered bymeans such as, but not limited to, injection, implantation, grafting,topical application, or parenterally.

The terms “apoptosis” or “programmed cell death” refer to a highlyregulated and active process that contributes to biologic homeostasiscomprised of a series of biochemical events that lead to a variety ofmorphological changes, including blebbing, changes to the cell membrane,such as loss of membrane asymmetry and attachment, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomal DNAfragmentation, without damaging the organism.

Apoptotic Pathways

Apoptotic cell death is induced by many different factors and involvesnumerous signaling pathways, some dependent on caspase proteases (aclass of cysteine proteases) and others that are caspase independent. Itcan be triggered by many different cellular stimuli, including cellsurface receptors, mitochondrial response to stress, and cytotoxic Tcells, resulting in activation of apoptotic signaling pathways.

The caspases involved in apoptosis convey the apoptotic signal in aproteolytic cascade, with caspases cleaving and activating othercaspases that then degrade other cellular targets that lead to celldeath. The caspases at the upper end of the cascade include caspase-8and caspase-9. Caspase-8 is the initial caspase involved in response toreceptors with a death domain (DD) like Fas.

Receptors in the TNF receptor family are associated with the inductionof apoptosis, as well as inflammatory signaling. The Fas receptor (CD95)mediates apoptotic signaling by Fas-ligand expressed on the surface ofother cells. The Fas-FasL interaction plays an important role in theimmune system and lack of this system leads to autoimmunity, indicatingthat Fas-mediated apoptosis removes self-reactive lymphocytes. Fassignaling also is involved in immune surveillance to remove transformedcells and virus infected cells. Binding of Fas to oligimerized FasL onanother cell activates apoptotic signaling through a cytoplasmic domaintermed the death domain (DD) that interacts with signaling adaptorsincluding FAF, FADD and DAX to activate the caspase proteolytic cascade.Caspase-8 and caspase-10 first are activated to then cleave and activatedownstream caspases and a variety of cellular substrates that lead tocell death.

Mitochondria participate in apoptotic signaling pathways through therelease of mitochondrial proteins into the cytoplasm. Cytochrome c, akey protein in electron transport, is released from mitochondria inresponse to apoptotic signals, and activates Apaf-1, a protease releasedfrom mitochondria. Activated Apaf-1 activates caspase-9 and the rest ofthe caspase pathway. Smac/DIABLO is released from mitochondria andinhibits IAP proteins that normally interact with caspase-9 to inhibitapoptosis. Apoptosis regulation by Bcl-2 family proteins occurs asfamily members form complexes that enter the mitochondrial membrane,regulating the release of cytochrome c and other proteins. TNF familyreceptors that cause apoptosis directly activate the caspase cascade,but can also activate Bid, a Bcl-2 family member, which activatesmitochondria-mediated apoptosis. Bax, another Bcl-2 family member, isactivated by this pathway to localize to the mitochondrial membrane andincrease its permeability, releasing cytochrome c and othermitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation,blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor)is a protein found in mitochondria that is released from mitochondria byapoptotic stimuli. While cytochrome C is linked to caspase-dependentapoptotic signaling, AIF release stimulates caspase-independentapoptosis, moving into the nucleus where it binds DNA. DNA binding byAIF stimulates chromatin condensation, and DNA fragmentation, perhapsthrough recruitment of nucleases.

The mitochondrial stress pathway begins with the release of cytochrome cfrom mitochondria, which then interacts with Apaf-1, causingself-cleavage and activation of caspase-9. Caspase-3, -6 and -7 aredownstream caspases that are activated by the upstream proteases and actthemselves to cleave cellular targets.

Granzyme B and perforin proteins released by cytotoxic T cells induceapoptosis in target cells, forming transmembrane pores, and triggeringapoptosis, perhaps through cleavage of caspases, althoughcaspase-independent mechanisms of Granzyme B mediated apoptosis havebeen suggested.

Fragmentation of the nuclear genome by multiple nucleases activated byapoptotic signaling pathways to create a nucleosomal ladder is acellular response characteristic of apoptosis. One nuclease involved inapoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse(CAD). DFF/CAD is activated through cleavage of its associated inhibitorICAD by caspases proteases during apoptosis. DFF/CAD interacts withchromatin components such as topoisomerase II and histone H1 to condensechromatin structure and perhaps recruit CAD to chromatin. Anotherapoptosis activated protease is endonuclease G (EndoG). EndoG is encodedin the nuclear genome but is localized to mitochondria in normal cells.EndoG may play a role in the replication of the mitochondrial genome, aswell as in apoptosis. Apoptotic signaling causes the release of EndoGfrom mitochondria. The EndoG and DFF/CAD pathways are independent sincethe EndoG pathway still occurs in cells lacking DFF.

Hypoxia, as well as hypoxia followed by reoxygenation, can triggercytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) aserine-threonine kinase ubiquitously expressed in most cell types,appears to mediate or potentiate apoptosis due to many stimuli thatactivate the mitochondrial cell death pathway. Loberg, R D, et al., J.Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated toinduce caspase 3 activation and to activate the proapoptotic tumorsuppressor gene p53. It also has been suggested that GSK-3 promotesactivation and translocation of the proapoptotic Bcl-2 family member,Bax, which, upon agregation and mitochondrial localization, inducescytochrome c release.

The term “attenuate” as used herein means to reduce the force, effect,or value of.

The terms “cardiac dilation” or “cardiac dilatation” are usedinterchangeably herein to refer to a condition where the size of theheart cavity becomes enlarged and stretched, thinning out the heartmuscle (myocardium).

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction. Theterms “chemotaxis” or “chemotactic” refer to the directed motion of amotile cell or part along a chemical concentration gradient towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “condition”, as used herein, refers to a variety of healthstates and is meant to include disorders or diseases caused by anyunderlying mechanism or injury.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells, which have a variety of effects on othercells. Cytokines mediate many important physiological functions,including growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins includinginterleukin 2 (IL-2), as well as several hematopoietic growth factors;type II cytokines, including the interferons and interleukin-10; tumornecrosis factor (“TNF”)-related molecules, including TNFα andlymphotoxin; immunoglobulin super-family members, including interleukin1 (“IL-1”); and the chemokines, a family of molecules that play acritical role in a wide variety of immune and inflammatory functions.The same cytokine can have different effects on a cell depending on thestate of the cell. Cytokines often regulate the expression of, andtrigger cascades of, other cytokines.

The term “disease” or “disorder,” as used herein, refers to animpairment of health or a condition of abnormal functioning.

The term “drug” as used herein refers to a therapeutic agent or anysubstance used in the prevention, diagnosis, alleviation, treatment, orcure of disease.

The term “echocardiography” as used herein refers to the use ofultrasound in the investigation of the heart and great vessels anddiagnosis of cardiovascular lesions. The term “echocardiogram as usedherein refers to the record obtained by echocardiography.

The term “ejection fraction” or “EF” as used herein refers to the amountof blood the left ventricle pumps out with each contraction. Theejection fraction is an important measurement in diagnosing and trackingheart failure. For example, a normal heart's ejection fraction may beequal to or greater than 55, whereas an EF measurement less than 40 maybe evidence of heart failure or cardiomyopathy. An EF between 40 and 55is indicative of damage, for example, from a previous heart attack. EFvalues between 45 and 54 are characterized as mildly abnormal; EF valuesbetween 30 and 44 are characterized as moderately abnormal; and EFvalues less than 30 are characterized as severely abnormal.

The term “end diastolic diameter” or “left ventricular diastolicdiameter” are used interchangeably herein to refer to the dimension ofthe left ventricle during the period in which the ventricles arerelaxing (i.e., diastole). Normal left ventricular diastolic diametervalues range, for example, from 3.9-5.3 for women and 4.2-5.9 for men.

The term “enhance” as used herein in its various grammatical formsrefers to an increase or to intensify in quality or quantity, or to makebetter or augment.

The term “enzymatic activity” as used herein refers to the action of anenzyme (meaning a protein that catalyzes a specific chemical reaction)on its target. It is quantified as the amount of substrate consumed (orproduct formed) in a given time under given conditions. The term“turnover number” as used herein refers to the number of molecules ofsubstrate that can be converted into product per catalytic site of agiven enzyme.

The term “fractional shortening” as used herein refers to a measure ofthe pump function of the heart. It is the ratio between the diameter ofthe left ventricle when it is relaxed and its diameter when it hascontracted. A normal value for fractional shortening is greater than26%.

The terms “functional equivalent” or “functionally equivalent” are usedinterchangeably herein to refer to substances, molecules,polynucleotides, proteins, peptides, or polypeptides having similar oridentical effects or use. A polypeptide functionally equivalent topolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1), for example, may havea biologic activity, e.g., an inhibitory activity, kinetic parameters,salt inhibition, a cofactor-dependent activity, and/or a functional unitsize that is substantially similar or identical to the expressedpolypeptide of SEQ ID NO: 1.

Examples of polypeptides functionally equivalent toYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 4),a polypeptide of amino acid sequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO:5), a polypeptide of amino acid sequence YARAAARQARAKALARQLAVA (SEQ IDNO: 6), a polypeptide of amino acid sequence YARAAARQARAKALARQLGVA (SEQID NO: 7), and a polypeptide of amino acid sequenceHRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 8).

The MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) peptide of aminoacid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) described in thepresent invention comprises a fusion protein in which a cell penetratingpeptide (CPP; YARAAARQARA; SEQ ID NO: 2) is operatively linked to atherapeutic domain (TD; KALARQLGVAA; SEQ ID NO: 3) in order to enhancetherapeutic efficacy.

Examples of polypeptides functionally equivalent to the therapeuticdomain (TD; KALARQLGVAA; SEQ ID NO: 3) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence KALARQLAVA (SEQ ID NO: 9), apolypeptide of amino acid sequence KALARQLGVA (SEQ ID NO: 10), apolypeptide of amino acid sequence KALARQLGVAA (SEQ ID NO: 11), apolypeptide of amino acid sequence KALNRQLGVAA (SEQ ID NO: 12), apolypeptide of amino acid sequence KAANRQLGVAA (SEQ ID NO: 13), apolypeptide of amino acid sequence KALNAQLGVAA (SEQ ID NO: 14), apolypeptide of amino acid sequence KALNRALGVAA (SEQ ID NO: 15), apolypeptide of amino acid sequence KALNRQAGVAA (SEQ ID NO: 16), apolypeptide of amino acid sequence KALNRQLAVA (SEQ ID NO: 17), apolypeptide of amino acid sequence KALNRQLAVAA (SEQ ID NO: 18), apolypeptide of amino acid sequence KALNRQLGAAA (SEQ ID NO: 19), and apolypeptide of amino acid sequence KALNRQLGVA (SEQ ID NO: 20).

Examples of polypeptides functionally equivalent to the cell penetratingpeptide (CPP; YARAAARQARA; SEQ ID NO: 2) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 21), apolypeptide of amino acid sequence WLRRIKA (SEQ ID NO: 22), apolypeptide of amino acid sequence YGRKKRRQRRR (SEQ ID NO: 23), apolypeptide of amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 24), apolypeptide of amino acid sequence FAKLAARLYR (SEQ ID NO: 25), apolypeptide of amino acid sequence KAFAKLAARLYR (SEQ ID NO: 26), and apolypeptide of amino acid sequence HRRIKAWLKKI (SEQ ID NO: 27).

The term “heterogeneous nuclear ribonucleoprotein AO” or “HNRNPA0” asused herein refers to a gene and the protein encoded therefrom thatbelongs to the A/B subfamily of ubiquitously expressed heterogeneousnuclear ribonucleoproteins (hnRNPs), which are RNA binding proteins thatcomplex with heterogeneous nuclear RNA (hnRNA). HNRNPA0 protein isassociated with pre-mRNAs in the nucleus and appear to influencepre-mRNA processing and other aspects of mRNA metabolism and transport.The HNRNPA0 protein encoded by the HNRNPA0 gene has two repeats ofquasi-RRM domains that bind RNAs, followed by a glycine-rich C-terminus.

The term “hypoxia” as used herein refers to a deficiency of oxygenreaching the tissues of the body; i.e., a condition in which tissues arestarved of oxygen. Hypoxia can lead, for example, to necrosis(cell/tissue death).

The term “immunomodulatory cell(s)” as used herein refer(s) to cell(s)that are capable of augmenting or diminishing immune responses byexpressing chemokines, cytokines and other mediators of immuneresponses.

The term “inflammatory cytokines” or “inflammatory mediators” as usedherein refers to the molecular mediators of the inflammatory process,which may modulate being either pro- or anti-inflamatory in theireffect. These soluble, diffusible molecules act both locally at the siteof tissue damage and infection and at more distant sites. Someinflammatory mediators are activated by the inflammatory process, whileothers are synthesized and/or released from cellular sources in responseto acute inflammation or by other soluble inflammatory mediators.Examples of inflammatory mediators of the inflammatory response include,but are not limited to, plasma proteases, complement, kinins, clottingand fibrinolytic proteins, lipid mediators, prostaglandins,leukotrienes, platelet-activating factor (PAF), peptides and amines,including, but not limited to, histamine, serotonin, and neuropeptides,pro-inflammatory cytokines, including, but not limited to,interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6),interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α),interferon-gamma (IF-γ), and interleukin-12 (IL-12).

The term “inhibit” and its various grammatical forms, including, but notlimited to, “inhibiting” or “inhibition”, are used herein to refer toreducing the amount or rate of a process, to stopping the processentirely, or to decreasing, limiting, or blocking the action or functionthereof. Inhibition can include a reduction or decrease of the amount,rate, action function, or process of a substance by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%.

The term “inhibitor” as used herein refers to a second molecule thatbinds to a first molecule thereby decreasing the first molecule'sactivity. Enzyme inhibitors are molecules that bind to enzymes therebydecreasing enzyme activity. The binding of an inhibitor can stop asubstrate from entering the active site of the enzyme and/or hinder theenzyme from catalyzing its reaction. Inhibitor binding is eitherreversible or irreversible. Irreversible inhibitors usually react withthe enzyme and change it chemically, for example, by modifying key aminoacid residues needed for enzymatic activity. In contrast, reversibleinhibitors bind non-covalently and produce different types of inhibitiondepending on whether these inhibitors bind the enzyme, theenzyme-substrate complex, or both. Enzyme inhibitors often are evaluatedby their specificity and potency.

The term “injury,” as used herein, refers to damage or harm to astructure or function of the body caused by an outside agent or force,which can be physical or chemical.

The term “interleukin (IL)” as used herein refers to a cytokine secretedby, and acting on, leukocytes. Interleukins regulate cell growth,differentiation, and motility, and stimulates immune responses, such asinflammation. Examples of interleukins include interleukin-1 (IL-1),interleukin 2 (IL-2), interleukin-la (IL-1α), interleukin-1β (IL-1β),interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) andinterleukin-12 (IL-12).

The term “interleukin-1” or “IL-1” as used herein refers to a cytokinederived primarily form mononuclear phagocytes, which enhances theproliferation of T helper cells and growth and differentiation of Bcells. When secreted in large quantities, IL-1 is a mediator ofinflammation, entering the bloodstream and causing fever, inducingsynthesis of acute phase proteins (including, but not limited to,ceruloplasmin, complement factor-3, haptoglobin, α-globulins,lipopolysaccharide binding protein, and the like) and initiatingmetabolic wasting. There are two distinct forms of interleukin-1, alpha(IL-1α) and beta (IL-1β), both of which perform the same functions, butrepresent different proteins.

The term “interleukin-6 or “IL-6” as used herein refers to a cytokinederived from macrophages and endothelial cells that increases synthesisand secretion of immunoglobulins by B lymphocytes. IL-6 also inducesacute phase proteins (including, but not limited to, ceruloplasmin,complement factor-3, haptoglobin, α-globulins, lipopolysaccharidebinding protein, and the like). In hepatocytes, IL-6 induces acute-phasereactants (including, but not limited to, erythrocyte sedimentation rate(ESR), C-reactive protein (CRP), fibrinogen, ferritin, and the like).

The term “interleukin-10” or “IL-10” as used herein refers to a cytokinederived from helper T cell lymphocytes (TH₂) that inhibitsgamma-interferon (IFN-γ) and IL-2 secretion by T cell lymphocytes (TH₁)and inhibits mononuclear cell inflammation.

The term “kinase” as used herein refers to a type of enzyme thattransfers phosphate groups from high-energy donor molecules to specifictarget molecules or substrates. High-energy donor groups can include,but are not limited, to GTP and ATP.

The term “left ventricular volume” are used herein to refers to thevolume of blood in the left ventricle of the heart. The term “leftventricular diastolic volume” as used herein refers to the volume ofblood in the left ventricle during the period in which the ventriclesare relaxing. Normal values range from 56-104 mL for women and 67-155 mLfor men. The term “left ventricular systolic volume” as used hereinrefers to the volume of blood in the left ventricle during the period inwhich the ventricles are contracting. Normal values range from 19-49 mLfor women and 22-58 mL for men.

The term “lymphocyte” refers to a small white blood cell formed inlymphatic tissue throughout the body and in normal adults making upabout 22-28% of the total number of leukocytes in the circulating bloodthat plays a large role in defending the body against disease.Individual lymphocytes are specialized in that they are committed torespond to a limited set of structurally related antigens. Thiscommitment, which exists before the first contact of the immune systemwith a given antigen, is expressed by the presence on the lymphocyte'ssurface membrane of receptors specific for determinants (epitopes) onthe antigen. Each lymphocyte possesses a population of receptors, all ofwhich have identical combining sites. One set, or clone, of lymphocytesdiffers from another clone in the structure of the combining region ofits receptors and thus differs in the epitopes that it can recognize.Lymphocytes differ from each other not only in the specificity of theirreceptors, but also in their functions.

Two broad classes of lymphocytes are recognized: the B-lymphocytes(B-cells), which are precursors of antibody-secreting cells, andT-lymphocytes (T-cells).

B-Lymphocytes

B-lymphocytes are derived from hematopoietic cells of the bone marrow. Amature B-cell can be activated with an antigen that expresses epitopesthat are recognized by its cell surface. The activation process may bedirect, dependent on cross-linkage of membrane Ig molecules by theantigen (cross-linkage-dependent B-cell activation), or indirect, viainteraction with a helper T-cell, in a process referred to as cognatehelp. In many physiological situations, receptor cross-linkage stimuliand cognate help synergize to yield more vigorous B-cell responses.(Paul, W. E., “Chapter 1: The immune system: an introduction,”Fundamental Immunology, 4^(th) Edition, Ed. Paul, W. E., Lippicott-RavenPublishers, Philadelphia (1999)).

Cross-linkage dependent B-cell activation requires that the antigenexpress multiple copies of the epitope complementary to the binding siteof the cell surface receptors because each B-cell expresses Ig moleculeswith identical variable regions. Such a requirement is fulfilled byother antigens with repetitive epitopes, such as capsularpolysaccharides of microorganisms or viral envelope proteins.Cross-linkage-dependent B-cell activation is a major protective immuneresponse mounted against these microbes. (Paul, W. E., “Chapter 1: Theimmune system: an introduction,” Fundamental Immunology, 4^(th) Edition,Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).

Cognate help allows B-cells to mount responses against antigens thatcannot cross-link receptors and, at the same time, providescostimulatory signals that rescue B cells from inactivation when theyare stimulated by weak cross-linkage events. Cognate help is dependenton the binding of antigen by the B-cell's membrane immunoglobulin (Ig),the endocytosis of the antigen, and its fragmentation into peptideswithin the endosomal/lysosomal compartment of the cell. Some of theresultant peptides are loaded into a groove in a specialized set of cellsurface proteins known as class II major histocompatibility complex(MHC) molecules. The resultant class II/peptide complexes are expressedon the cell surface and act as ligands for the antigen-specificreceptors of a set of T-cells designated as CD4+ T-cells. The CD4+T-cells bear receptors on their surface specific for the B-cell's classII/peptide complex. B-cell activation depends not only on the binding ofthe T cell through its T cell receptor (TCR), but this interaction alsoallows an activation ligand on the T-cell (CD40 ligand) to bind to itsreceptor on the B-cell (CD40) signaling B-cell activation. In addition,T helper cells secrete several cytokines that regulate the growth anddifferentiation of the stimulated B-cell by binding to cytokinereceptors on the B cell. (Paul, W. E., “Chapter 1: The immune system: anintroduction,” Fundamental Immunology, 4^(th) Edition, Ed. Paul, W. E.,Lippicott-Raven Publishers, Philadelphia (1999)).

During cognate help for antibody production, the CD40 ligand istransiently expressed on activated CD4+T helper cells, and it binds toCD40 on the antigen-specific B cells, thereby tranducing a secondcostimulatory signal. The latter signal is essential for B cell growthand differentiation and for the generation of memory B cells bypreventing apoptosis of germinal center B cells that have encounteredantigen. Hyperexpression of the CD40 ligand in both B and T cells isimplicated in the pathogenic autoantibody production in human SLEpatients. (Desai-Mehta, A. et al., “Hyperexpression of CD40 ligand by Band T cells in human lupus and its role in pathogenic autoantibodyproduction,” J. Clin. Invest., 97(9): 2063-2073 (1996)).

T-Lymphocytes

T-lymphocytes derive from precursors in hematopoietic tissue, undergodifferentiation in the thymus, and are then seeded to peripherallymphoid tissue and to the recirculating pool of lymphocytes.T-lymphocytes or T cells mediate a wide range of immunologic functions.These include the capacity to help B cells develop intoantibody-producing cells, the capacity to increase the microbicidalaction of monocytes/macrophages, the inhibition of certain types ofimmune responses, direct killing of target cells, and mobilization ofthe inflammatory response. These effects depend on their expression ofspecific cell surface molecules and the secretion of cytokines. (Paul,W. E., “Chapter 1: The immune system: an introduction,” FundamentalImmunology, 4^(th) Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

T cells differ from B cells in their mechanism of antigen recognition.Immunoglobulin, the B cell's receptor, binds to individual epitopes onsoluble molecules or on particulate surfaces. B-cell receptors seeepitopes expressed on the surface of native molecules. Antibody andB-cell receptors evolved to bind to and to protect againstmicroorganisms in extracellular fluids. In contrast, T cells recognizeantigens on the surface of other cells and mediate their functions byinteracting with, and altering, the behavior of these antigen-presentingcells (APCs). There are three main types of antigen-presenting cells inperipheral lymphoid organs that can activate T cells: dendritic cells,macrophages and B cells. The most potent of these are the dendriticcells, whose only function is to present foreign antigens to T cells.Immature dendritic cells are located in tissues throughout the body,including the skin, gut, and respiratory tract. When they encounterinvading microbes at these sites, they endocytose the pathogens andtheir products, and carry them via the lymph to local lymph nodes or gutassociated lymphoid organs. The encounter with a pathogen induces thedendritic cell to mature from an antigen-capturing cell to anantigen-presenting cell (APC) that can activate T cells. APCs displaythree types of protein molecules on their surface that have a role inactivating a T cell to become an effector cell: (1) MHC proteins, whichpresent foreign antigen to the T cell receptor; (2) costimulatoryproteins which bind to complementary receptors on the T cell surface;and (3) cell-cell adhesion molecules, which enable a T cell to bind tothe antigen-presenting cell (APC) for long enough to become activated.(“Chapter 24: The adaptive immune system,” Molecular Biology of theCell, Alberts, B. et al., Garland Science, N Y, 2002).

T-cells are subdivided into two distinct classes based on the cellsurface receptors they express. The majority of T cells express T cellreceptors (TCR) consisting of c and 3 chains. A small group of T cellsexpress receptors made of γ and δ chains. Among the α/β T cells are twoimportant sublineages: those that express the coreceptor molecule CD4(CD4+ T cells); and those that express CD8 (CD8+ T cells). These cellsdiffer in how they recognize antigen and in their effector andregulatory functions.

CD4+ T cells are the major regulatory cells of the immune system. Theirregulatory function depends both on the expression of their cell-surfacemolecules, such as CD40 ligand whose expression is induced when the Tcells are activated, and the wide array of cytokines they secrete whenactivated.

T cells also mediate important effector functions, some of which aredetermined by the patterns of cytokines they secrete. The cytokines canbe directly toxic to target cells and can mobilize potent inflammatorymechanisms.

In addition, T cells particularly CD8+ T cells, can develop intocytotoxic T-lymphocytes (CTLs) capable of efficiently lysing targetcells that express antigens recognized by the CTLs. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology,4^(th) Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

T cell receptors (TCRs) recognize a complex consisting of a peptidederived by proteolysis of the antigen bound to a specialized groove of aclass II or class I MHC protein. The CD4+ T cells recognize onlypeptide/class II complexes while the CD8+ T cells recognizepeptide/class I complexes. (Paul, W. E., “Chapter 1: The immune system:an introduction,” Fundamental Immunology, 4^(th) Edition, Ed. Paul, W.E., Lippicott-Raven Publishers, Philadelphia (1999)).

The TCR's ligand (i.e., the peptide/MHC protein complex) is createdwithin antigen-presenting cells (APCs). In general, class II MHCmolecules bind peptides derived from proteins that have been taken up bythe APC through an endocytic process. These peptide-loaded class IImolecules are then expressed on the surface of the cell, where they areavailable to be bound by CD4+ T cells with TCRs capable of recognizingthe expressed cell surface complex. Thus, CD4+ T cells are specializedto react with antigens derived from extracellular sources. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology,4^(th) Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

In contrast, class I MHC molecules are mainly loaded with peptidesderived from internally synthesized proteins, such as viral proteins.These peptides are produced from cytosolic proteins by proteolysis bythe proteosome and are translocated into the rough endoplasmicreticulum. Such peptides, generally nine amino acids in length, arebound into the class I MHC molecules and are brought to the cellsurface, where they can be recognized by CD8+ T cells expressingappropriate receptors. This gives the T cell system, particularly CD8+ Tcells, the ability to detect cells expressing proteins that aredifferent from, or produced in much larger amounts than, those of cellsof the remainder of the organism (e.g., vial antigens) or mutantantigens (such as active oncogene products), even if these proteins intheir intact form are neither expressed on the cell surface norsecreted. (Paul, W. E., “Chapter 1: The immune system: an introduction,”Fundamental Immunology, 4^(th) Edition, Ed. Paul, W. E., Lippicott-RavenPublishers, Philadelphia (1999)).

T cells can also be classified based on their function as helper Tcells; T cells involved in inducing cellular immunity; suppressor Tcells; and cytotoxic T cells.

Helper T Cells

Helper T cells are T cells that stimulate B cells to make antibodyresponses to proteins and other T cell-dependent antigens. Tcell-dependent antigens are immunogens in which individual epitopesappear only once or a limited number of times such that they are unableto cross-link the membrane immunoglobulin (Ig) of B cells or do soinefficiently. B cells bind the antigen through their membrane Ig, andthe complex undergoes endocytosis. Within the endosomal and lysosomalcompartments, the antigen is fragmented into peptides by proteolyticenzymes and one or more of the generated peptides are loaded into classII MHC molecules, which traffic through this vesicular compartment. Theresulting peptide/class II MHC complex is then exported to the B-cellsurface membrane. T cells with receptors specific for the peptide/classII molecular complex recognize this complex on the B-cell surface.(Paul, W. E., “Chapter 1: The immune system: an introduction,”Fundamental Immunology, 4^(th) Edition, Ed. Paul, W. E., Lippicott-RavenPublishers, Philadelphia (1999)).

B-cell activation depends both on the binding of the T cell through itsTCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40on the B cell. T cells do not constitutively express CD40L. Rather,CD40L expression is induced as a result of an interaction with an APCthat expresses both a cognate antigen recognized by the TCR of the Tcell and CD80 or CD86. CD80/CD86 is generally expressed by activated,but not resting, B cells so that the helper interaction involving anactivated B cell and a T cell can lead to efficient antibody production.In many cases, however, the initial induction of CD40L on T cells isdependent on their recognition of antigen on the surface of APCs thatconstitutively express CD80/86, such as dendritic cells. Such activatedhelper T cells can then efficiently interact with and help B cells.Cross-linkage of membrane Ig on the B cell, even if inefficient, maysynergize with the CD40L/CD40 interaction to yield vigorous B-cellactivation. The subsequent events in the B-cell response, includingproliferation, Ig secretion, and class switching (of the Ig class beingexpressed) either depend or are enhanced by the actions of Tcell-derived cytokines. (Paul, W. E., “Chapter 1: The immune system: anintroduction,” Fundamental Immunology, 4^(th) Edition, Ed. Paul, W. E.,Lippicott-Raven Publishers, Philadelphia (1999)).

CD4+ T cells tend to differentiate into cells that principally secretethe cytokines IL-4, IL-5, IL-6, and IL-10 (T_(H2) cells) or into cellsthat mainly produce IL-2, IFN-γ, and lymphotoxin (T_(H1) cells). TheT_(H2) cells are very effective in helping B-cells develop intoantibody-producing cells, whereas the T_(H1) cells are effectiveinducers of cellular immune responses, involving enhancement ofmicrobicidal activity of monocytes and macrophages, and consequentincreased efficiency in lysing microorganisms in intracellular vesicularcompartments. Although the CD4+ T cells with the phenotype of T_(H2)cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells,T_(H1) cells also have the capacity to be helpers. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology,4^(th) Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,Philadelphia (1999)).

T Cells Involved in Induction of Cellular Immunity

T cells also may act to enhance the capacity of monocytes andmacrophages to destroy intracellular microorganisms. In particular,interferon-gamma (IFN-γ) produced by helper T cells enhances severalmechanisms through which mononuclear phagocytes destroy intracellularbacteria and parasitism including the generation of nitric oxide andinduction of tumor necrosis factor (TNF) production. The T_(H1) cellsare effective in enhancing the microbicidal action because they produceIFN-γ. By contrast, two of the major cytokines produced by T_(H2) cells,IL-4 and IL-10, block these activities. (Paul, W. E., “Chapter 1: Theimmune system: an introduction,” Fundamental Immunology, 4^(th) Edition,Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).

Suppressor or Regulatory T (Treg) Cells

A controlled balance between initiation and downregulation of the immuneresponse is important to maintain immune homeostasis. Both apoptosis andT cell anergy (a tolerance mechanism in which the T cells areintrinsically functionally inactivated following an antigen encounter(Scwartz, R. H., “T cell anergy,” Annu. Rev. Immunol., 21: 305-334(2003)) are important mechanisms that contribute to the downregulationof the immune response. A third mechanism is provided by activesuppression of activated T cells by suppressor or regulatory CD4+T(Treg) cells. (Reviewed in Kronenberg, M. et al., “Regulation ofimmunity by self-reactive T cells,” Nature 435: 598-604 (2005)). CD4+Tregs that constitutively express the IL-2 receptor alpha (IL-2Rα) chain(CD4+CD25+) are a naturally occurring T cell subset that are anergic andsuppressive. (Taams, L. S. et 1., “Human anergic/suppressive CD4+CD25+ Tcells: a highly differentiated and apoptosis-prone population,” Eur. J.Immunol., 31: 1122-1131 (2001)). Depletion of CD4⁺CD25⁺ Tregs results insystemic autoimmune disease in mice. Furthermore, transfer of theseTregs prevents development of autoimmune disease. Human CD4⁺CD25⁺ Tregs,similar to their murine counterpart, are generated in the thymus and arecharacterized by the ability to suppress proliferation of responder Tcells through a cell-cell contact-dependent mechanism, the inability toproduce IL-2, and the anergic phenotype in vitro. Human CD4⁺CD25⁺ Tcells can be split into suppressive (CD25^(high)) and nonsuppressive(CD25^(low)) cells, according to the level of CD25 expression. A memberof the forkhead family of transcription factors, FOXP3, has been shownto be expressed in murine and human CD4⁺CD25⁺ Tregs and appears to be amaster gene controlling CD4⁺CD25⁺ Treg development. (Battaglia, M. etal., “Rapamycin promotes expansion of functional CD4+CD25+Foxp3+regulator T cells of both healthy subjects and type 1 diabeticpatients,” J. Immunol., 177: 8338-8347 (200)).

Cytotoxic T Lymphocytes (CTL)

The CD8+ T cells that recognize peptides from proteins produced withinthe target cell have cytotoxic properties in that they lead to lysis ofthe target cells. The mechanism of CTL-induced lysis involves theproduction by the CTL of perforin, a molecule that can insert into themembrane of target cells and promote the lysis of that cell.Perforin-mediated lysis is enhanced by a series of enzymes produced byactivated CTLs, referred to as granzymes. Many active CTLs also expresslarge amounts of fas ligand on their surface. The interaction of fasligand on the surface of CTL with fas on the surface of the target cellinitiates apoptosis in the target cell, leading to the death of thesecells. CTL-mediated lysis appears to be a major mechanism for thedestruction of virally infected cells.

The term “lactate dehydrogenase” or “LDH” as used herein refers to agroup of enzymes which include, but are not limited to, L-1dehydrogenase and D-1 dehydrogenase. LDH functions include, but are notlimited to, transfer of hydrogen to ferrictytochrome c or to cytochromeb2, transfer of hydrogen to NAD⁺, and the like. An increased amount ofLDH in the blood may be a sign of tissue damage (e.g., heart tissuedamage).

The term “macrophage inflammatory protein 1” or “MIP1” as used hereinrefers to a cytokine composed of several gene products that has beenidentified in activated T cells, macrophages, and fibroblasts. MIP1exerts an effect, for example, on neutrophils, monocytes andhematopoietic cells and its functions include, but are not limited to,regulating inflammation and cell growth.

The term “macrophage inflammatory protein 2” or “MIP2” as used hereinrefers to platelet products including, but not limited to, plateletfactor 4 and 3-thromboglobulin that have effects on a number of celltypes including, but not limited to, neutrophils, fibroblasts,hematopoietic cells and melanoma cells. MIP2 functions include, but arenot limited to, regulating inflammation and cell growth.

The term “MK2i peptide” or “MK2i” or “MMI-0100” as used interchangeablyherein refers to a peptide of amino acid sequence YARAAARQARAKALARQLGVAA(SEQ ID NO: 1) comprising a fusion protein in which a proteintransduction domain (PTD; YARAAARQARA; SEQ ID NO: 2) is operativelylinked to a therapeutic domain (KALARQLGVAA; SEQ ID NO: 3).

The term “MK2 kinase” or “MK2” as used herein refers tomitogen-activated protein kinase-activated protein kinase 2 (alsoreferred to as “MAPKAPK2”, “MAPKAP-K2”, “MK2”), which is a member of theserine/threonine (Ser/Thr) protein kinase family.

The term “modify” as used herein means to change, vary, adjust, temper,alter, affect or regulate to a certain measure or proportion in one ormore particulars.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “normoxia” as used herein refers to a normal level of oxygen; anormal oxygen state.

The term “necrosis” refers to the premature death of cells and livingtissue induced by external factors, such as infection, toxins or trauma.Necrotic tissue undergoes chemical reactions different from those ofapoptotic tissue. Necrosis typically begins with cell swelling,chromatin digestion, disruption of the plasma membrane and of organellemembranes. Damage to the lysosome membrane can trigger release oflysosomal enzymes, destroying other parts of the cell. Late necrosis ischaracterized by extensive DNA hydrolysis, vacuolation of theendoplasmic reticulum, organelle breakdown and cell lysis. The releaseof intracellular content after plasma membrane rupture is the cause ofinflammation in necrosis. Released lysosomal enzymes can trigger a chainreaction of further cell death. Necrosis of a sufficient amount ofcontiguous tissue can result in tissue death or gangrene.

The term “neutrophils” or “polymorphonuclear neutrophils (PMNs)” as usedherein refers to the most abundant type of white blood cells in mammals,which form an essential part of the innate immune system. They form partof the polymorphonuclear cell family (PMNs) together with basophils andeosinophils. Neutrophils are normally found in the blood stream. Duringthe beginning (acute) phase of inflammation, particularly as a result ofbacterial infection and some cancers, neutrophils are one of thefirst-responders of inflammatory cells to migrate toward the site ofinflammation. They migrate through the blood vessels, then throughinterstitial tissue, following chemical signals such as interleukin-8(IL-8) and C5a in a process called chemotaxis, the directed motion of amotile cell or part along a chemical concentration gradient towardenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “nucleic acid” is used herein to refer to a deoxyribonucleotideor ribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

The term “nucleotide” is used herein to refer to a chemical compoundthat consists of a heterocyclic base, a sugar, and one or more phosphategroups. In the most common nucleotides, the base is a derivative ofpurine or pyrimidine, and the sugar is the pentose deoxyribose orribose. Nucleotides are the monomers of nucleic acids, with three ormore bonding together in order to form a nucleic acid. Nucleotides arethe structural units of RNA, DNA, and several cofactors, including, butnot limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine(A), and guanine (G); pyrimidines include cytosine (C), thymine (T), anduracil (U).

The following terms are used herein to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity.”

(a) The term “reference sequence” refers to a sequence used as a basisfor sequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) The term “comparison window” refers to a contiguous and specifiedsegment of a polynucleotide sequence, wherein the polynucleotidesequence may be compared to a reference sequence and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be at least 30contiguous nucleotides in length, at least 40 contiguous nucleotides inlength, at least 50 contiguous nucleotides in length, at least 100contiguous nucleotides in length, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence, a gap penaltytypically is introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences,8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology,24:307-331 (1994). The BLAST family of programs, which can be used fordatabase similarity searches, includes: BLASTN for nucleotide querysequences against nucleotide database sequences; BLASTX for nucleotidequery sequences against protein database sequences; BLASTP for proteinquery sequences against protein database sequences; TBLASTN for proteinquery sequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters. Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center forBiotechnology-Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits then are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a word length (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins may be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats, or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs may be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17:191-201 (1993)) low-complexity filters may beemployed alone or in combination.

(c) The term “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences is used herein to refer to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions that are not identical often differ by conservativeamino acid substitutions, i.e., where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

(d) The term “percentage of sequence identity” is used herein mean thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, at least 80% sequence identity, at least 90% sequenceidentity and at least 95% sequence identity, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values may beadjusted appropriately to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, or at least 70%, atleast 80%, at least 90%, or at least 95%. Another indication thatnucleotide sequences are substantially identical is if two moleculeshybridize to each other under stringent conditions. However, nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides that they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide that the first nucleicacid encodes is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

As used herein, the terms “oral” or “orally” refer to the introductioninto the body by mouth whereby absorption occurs in one or more of thefollowing areas of the body: the mouth, stomach, small intestine, lungs(also specifically referred to as inhalation), and the small bloodvessels under the tongue (also specifically referred to assublingually). The term “pharmaceutical composition” as used hereinrefers to a preparation comprising a pharmaceutical product, drug,metabolite, or active ingredient.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle); intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord), intrasternal injection, or infusiontechniques. A parenterally administered composition of the describedinvention is delivered using a needle, e.g., a surgical needle. The term“surgical needle” as used herein, refers to any needle adapted fordelivery of fluid (i.e., capable of flow) compositions of the describedinvention into a selected anatomical structure. Injectable preparations,such as sterile injectable aqueous or oleaginous suspensions, can beformulated according to the known art using suitable dispersing orwetting agents and suspending agents.

As used herein the term “pharmaceutically acceptable carrier” refers toany substantially non-toxic carrier conventionally useable foradministration of pharmaceuticals in which the isolated polypeptide ofthe present invention will remain stable and bioavailable. Thepharmaceutically acceptable carrier must be of sufficiently high purityand of sufficiently low toxicity to render it suitable foradministration to the mammal being treated. It further should maintainthe stability and bioavailability of an active agent. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition.

The term “pharmaceutically acceptable salt” means those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio.

The term “phosphorylated-MK2 kinase”, “phospho-MK2” or “p-MK2” as usedherein refers to phosphorylated mitogen-activated proteinkinase-activated protein kinase 2 (also referred to as “MAPKAPK2”,“MAPKAP-K2”, “MK2”), which is a member of the serine/threonine (Ser/Thr)protein kinase family.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids.

The terms “polypeptide” and “protein” also are used herein in theirbroadest sense to refer to a sequence of subunit amino acids, amino acidanalogs, or peptidomimetics. The subunits are linked by peptide bonds,except where noted. The polypeptides described herein may be chemicallysynthesized or recombinantly expressed. Polypeptides of the describedinvention also can be synthesized chemically. Synthetic polypeptides,prepared using the well-known techniques of solid phase, liquid phase,or peptide condensation techniques, or any combination thereof, caninclude natural and unnatural amino acids. Amino acids used for peptidesynthesis may be standard Boc (N-α-amino protectedN-α-t-butyloxycarbonyl) amino acid resin with the standard deprotecting,neutralization, coupling and wash protocols of the original solid phaseprocedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or thebase-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) aminoacids first described by Carpino and Han (1972, J. Org. Chem.37:3403-3409). Both Fmoc and Boc N-α-amino protected amino acids can beobtained from Sigma, Cambridge Research Biochemical, or other chemicalcompanies familiar to those skilled in the art. In addition, thepolypeptides can be synthesized with other N-α-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, for example, in Stewart and Young, 1984, Solid PhaseSynthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fieldsand Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or usingautomated synthesizers. The polypeptides of the invention may compriseD-amino acids (which are resistant to L-amino acid-specific proteases invivo), a combination of D- and L-amino acids, and various “designer”amino acids (e.g., β-methyl amino acids, C-α-methyl amino acids, andN-α-methyl amino acids, etc.) to convey special properties. Syntheticamino acids include ornithine for lysine, and norleucine for leucine orisoleucine. In addition, the polypeptides can have peptidomimetic bonds,such as ester bonds, to prepare peptides with novel properties. Forexample, a peptide may be generated that incorporates a reduced peptidebond, i.e., R1-CH₂—NH—R2, where R1 and R2 are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a polypeptide would be resistant to protease activity, andwould possess an extended half-live in vivo. Accordingly, these termsalso apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, the protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids.

The terms “polypeptide”, “peptide” and “protein” also are inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation, and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides may not be entirely linear.For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of posttranslational events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. In some embodiments, the peptideis of any length or size.

The terms “preserve”, “preserved”, “preserving” or “preservation” asused herein refer to maintaining, keeping safe from harm or injury,protecting, sparing or maintaining function.

The terms “prevent”, “prevented”, “preventing” or “prevention” as usedherein refer to the keeping, hindering or averting of an event, act, oraction from happening, occurring or arising.

The term “reduce” or “reducing” as used herein refers to the limiting ofan occurrence of a disorder in individuals at risk of developing thedisorder.

The term “similar” is used interchangeably with the terms analogous,comparable, or resembling, meaning having traits or characteristics incommon.

The term “solution” as used herein refers to a homogeneous mixture oftwo or more substances. It is frequently, though not necessarily, aliquid. In a solution, the molecules of the solute (or dissolvedsubstance) are uniformly distributed among those of the solvent.

The terms “soluble” and “solubility” refer to the property of beingsusceptible to being dissolved in a specified fluid (solvent). The term“insoluble” refers to the property of a material that has minimal orlimited solubility in a specified solvent. In a solution, the moleculesof the solute (or dissolved substance) are uniformly distributed amongthose of the solvent.

The term “stimulate” in any of its grammatical forms as used hereinrefers to inducing activation or increasing activity.

The term “suspension” as used herein refers to a dispersion (mixture) inwhich a finely-divided species is combined with another species, withthe former being so finely divided and mixed that it doesn't rapidlysettle out. In everyday life, the most common suspensions are those ofsolids in liquid.

As used herein, the terms “subject” or “individual” or “patient” areused interchangeably to refer to a member of an animal species ofmammalian origin, including humans. The term “a subject in need thereof”is used to refer to a subject having, or at risk of progression to heartfailure, including a subject having an AMI that leads to a diseasemanifestation of left ventricular remodeling.

The phrase “subject in need of such treatment” as used herein refers toa patient who suffers from a disease, disorder, condition, orpathological process. In some embodiments, the term “subject in need ofsuch treatment” also is used to refer to a patient who (i) will beadministered at least one polypeptide of the invention; (ii) isreceiving at least one polypeptide of the invention; or (iii) hasreceived at least one polypeptide of the invention, unless the contextand usage of the phrase indicates otherwise.

The term “substantially similar” as used herein means that a firstvalue, aspect, trait, feature, number, or amount is of at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofa second value, aspect, trait, feature, number, or amount. For example,polypeptide substantially similar to MMI-0100 (YARAAARQARAKALARQLGVAA(SEQ ID NO: 1)) would have at least 70% amino acid sequence identity, atleast 75% amino acid sequence identity, at least 80% amino acid sequenceidentity, at least 90% sequence identity, or at least 95% amino acidsequence identity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1).

The term “substitution” is used herein to refer to a situation in whicha base or bases are exchanged for another base or bases in a DNAsequence. Substitutions may be synonymous substitutions or nonsynonymoussubstitutions. As used herein, “synonymous substitutions” refer tosubstitutions of one base for another in an exon of a gene coding for aprotein, such that the amino acid sequence produced is not modified. Theterm “nonsynonymous substitutions” as used herein refer to substitutionsof one base for another in an exon of a gene coding for a protein, suchthat the amino acid sequence produced is modified.

The term “symptom” as used herein refers to a phenomenon that arisesfrom and accompanies a particular disease or disorder and serves as anindication of it.

The term “syndrome,” as used herein, refers to a pattern of symptomsindicative of some disease or condition.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, metabolite, composition or other substance thatprovides a therapeutic effect. The term “active” as used herein refersto the ingredient, component or constituent of the compositions of thedescribed invention responsible for the intended therapeutic effect. Theterms “therapeutic agent” and “active agent” are used interchangeablyherein. The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e., dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED50 whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The terms “therapeutic amount”, “therapeutically effective amount”, an“amount effective”, or “pharmaceutically effective amount” of an activeagent is used interchangeably to refer to an amount that is sufficientto provide the intended benefit of treatment. An effective amount of theactive agent(s) that can be employed according to the describedinvention generally ranges from about 0.25 mg/kg body weight to about160 mg/kg body weight per dose, with three doses given per day. However,dosage levels are based on a variety of factors, including the type ofinjury, the age, weight, sex, medical condition of the patient, theseverity of the condition, the route of administration, and theparticular active agent employed. Thus the dosage regimen may varywidely, but can be determined routinely by a physician using standardmethods. Additionally, the terms “therapeutic amount”, “therapeuticallyeffective amount” and “pharmaceutically effective amount” includesprophylactic or preventative amounts of the compositions of thedescribed invention. In prophylactic or preventative applications of thedescribed invention, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk of, adisease, disorder or condition in an amount sufficient to eliminate orreduce the risk, lessen the severity, or delay the onset of the disease,disorder or condition, including biochemical, histologic and/orbehavioral symptoms of the disease, disorder or condition, itscomplications, and intermediate pathological phenotypes presentingduring development of the disease, disorder or condition. It isgenerally preferred that a maximum dose be used, that is, the highestsafe dose according to some medical judgment. The terms “dose” and“dosage” are used interchangeably herein.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect can include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect can also include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

For any therapeutic agent described herein the therapeutically effectiveamount may be initially determined from preliminary in vitro studiesand/or animal models. A therapeutically effective dose may also bedetermined from human data. The applied dose may be adjusted based onthe relative bioavailability and potency of the administered compound.Adjusting the dose to achieve maximal efficacy based on the methodsdescribed above and other well-known methods is within the capabilitiesof the ordinarily skilled artisan.

General principles for determining therapeutic effectiveness, which maybe found in Chapter 1 of Goodman and Gilman's The Pharmacological Basisof Therapeutics, 10th Edition, McGraw-Hill (New York) (2001),incorporated herein by reference, are summarized below.

Pharmacokinetic principles provide a basis for modifying a dosageregimen to obtain a desired degree of therapeutic efficacy with aminimum of unacceptable adverse effects. In situations where the drug'splasma concentration can be measured and related to the therapeuticwindow, additional guidance for dosage modification can be obtained.

Drug products are considered to be pharmaceutical equivalents if theycontain the same active ingredients and are identical in strength orconcentration, dosage form, and route of administration. Twopharmaceutically equivalent drug products are considered to bebioequivalent when the rates and extents of bioavailability of theactive ingredient in the two products are not significantly differentunder suitable test conditions.

The term “therapeutic window” refers to a concentration range thatprovides therapeutic efficacy without unacceptable toxicity. Followingadministration of a dose of a drug, its effects usually show acharacteristic temporal pattern. A lag period is present before the drugconcentration exceeds the minimum effective concentration (“MEC”) forthe desired effect. Following onset of the response, the intensity ofthe effect increases as the drug continues to be absorbed anddistributed. This reaches a peak, after which drug elimination resultsin a decline in the effect's intensity that disappears when the drugconcentration falls back below the MEC. Accordingly, the duration of adrug's action is determined by the time period over which concentrationsexceed the MEC. The therapeutic goal is to obtain and maintainconcentrations within the therapeutic window for the desired responsewith a minimum of toxicity. Drug response below the MEC for the desiredeffect will be subtherapeutic, whereas for an adverse effect, theprobability of toxicity will increase above the MEC. Increasing ordecreasing drug dosage shifts the response curve up or down theintensity scale and is used to modulate the drug's effect. Increasingthe dose also prolongs a drug's duration of action but at the risk ofincreasing the likelihood of adverse effects. Accordingly, unless thedrug is nontoxic, increasing the dose is not a useful strategy forextending a drug's duration of action.

Instead, another dose of drug should be given to maintain concentrationswithin the therapeutic window. In general, the lower limit of thetherapeutic range of a drug appears to be approximately equal to thedrug concentration that produces about half of the greatest possibletherapeutic effect, and the upper limit of the therapeutic range is suchthat no more than about 5% to about 10% of patients will experience atoxic effect. These figures can be highly variable, and some patientsmay benefit greatly from drug concentrations that exceed the therapeuticrange, while others may suffer significant toxicity at much lowervalues. The therapeutic goal is to maintain steady-state drug levelswithin the therapeutic window. For most drugs, the actual concentrationsassociated with this desired range are not and need not be known, and itis sufficient to understand that efficacy and toxicity are generallyconcentration-dependent, and how drug dosage and frequency ofadministration affect the drug level. For a small number of drugs wherethere is a small (two- to three-fold) difference between concentrationsresulting in efficacy and toxicity, a plasma-concentration rangeassociated with effective therapy has been defined.

In this case, a target level strategy is reasonable, wherein a desiredtarget steady-state concentration of the drug (usually in plasma)associated with efficacy and minimal toxicity is chosen, and a dosage iscomputed that is expected to achieve this value. Drug concentrationssubsequently are measured and dosage is adjusted if necessary toapproximate the target more closely.

In most clinical situations, drugs are administered in a series ofrepetitive doses or as a continuous infusion to maintain a steady-stateconcentration of drug associated with the therapeutic window. Tomaintain the chosen steady-state or target concentration (“maintenancedose”), the rate of drug administration is adjusted such that the rateof input equals the rate of loss. If the clinician chooses the desiredconcentration of drug in plasma and knows the clearance andbioavailability for that drug in a particular patient, the appropriatedose and dosing interval can be calculated.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The term “tumor necrosis factor alpha (a)” or “TNF-α” (also known ascachectin and TNFSF2) as used herein refers to a pleiotropic moleculethat plays a central role in inflammation, apoptosis, and immune systemdevelopment. TNF-α is produced by a wide variety of immune andepithelial cell types. Human TNF-α consists of a 35 amino acidcytoplasmic domain, a 21 amino acid transmembrane segment, and a 177amino acid extracellular domain. Cleavage of membrane bound TNF-α byTACE/ADAM17 releases a 55 kDa soluble trimeric form of TNF-α. TNF-αtrimers bind the ubiquitous TNF RI and the hematopoietic cell-restrictedTNF RII, both of which are also expressed as homotrimers. TNF-αregulates lymphoid tissue development through control of apoptosis. Italso promotes inflammatory responses by inducing the activation ofvascular endothelial cells and macrophages. TNF-α is a key cytokine inthe development of several inflammatory disorders. It contributes to thedevelopment of type 2 diabetes through its effects on insulin resistanceand fatty acid metabolism.

The terms “variants”, “mutants”, and “derivatives” are used herein torefer to nucleotide or polypeptide sequences with substantial identityto a reference nucleotide or polypeptide sequence. The differences inthe sequences may be the result of changes, either naturally or bydesign, in sequence or structure. Natural changes may arise during thecourse of normal replication or duplication in nature of the particularnucleic acid sequence. Designed changes may be specifically designed andintroduced into the sequence for specific purposes. Such specificchanges may be made in vitro using a variety of mutagenesis techniques.Such sequence variants generated specifically may be referred to as“mutants” or “derivatives” of the original sequence.

A skilled artisan likewise can produce polypeptide variants ofpolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) having single ormultiple amino acid substitutions, deletions, additions or replacements,but functionally equivalent to SEQ ID NO: 1. These variants may includeinter alia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids; (b)variants in which one or more amino acids are added; (c) variants inwhich at least one amino acid includes a substituent group; (d) variantsin which amino acid residues from one species are substituted for thecorresponding residue in another species, either at conserved ornon-conserved positions; and (d) variants in which a target protein isfused with another peptide or polypeptide such as a fusion partner, aprotein tag or other chemical moiety, that may confer useful propertiesto the target protein, for example, an epitope for an antibody. Thetechniques for obtaining such variants, including, but not limited to,genetic (suppressions, deletions, mutations, etc.), chemical, andenzymatic techniques, are known to the skilled artisan. As used herein,the term “mutation” refers to a change of the DNA sequence within a geneor chromosome of an organism resulting in the creation of a newcharacter or trait not found in the parental type, or the process bywhich such a change occurs in a chromosome, either through an alterationin the nucleotide sequence of the DNA coding for a gene or through achange in the physical arrangement of a chromosome. Three mechanisms ofmutation include substitution (exchange of one base pair for another),addition (the insertion of one or more bases into a sequence), anddeletion (loss of one or more base pairs).

The term “vehicle” as used herein refers to a substance that facilitatesthe use of a drug or other material that is mixed with it.

According to one embodiment, the described invention provides apharmaceutical composition comprising a therapeutic amount of aMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)inhibitor comprising an MK2 polypeptide inhibitor or a functionalequivalent thereof, and a pharmaceutically acceptable carrier. Accordingto another embodiment, the MK2 polypeptide inhibitor is a polypeptideMMI-0100 of amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to one embodiment, the described invention provides a kitcomprising a pharmaceutical composition and a packaging material.According to another embodiment, the kit further comprises a means foradministering the pharmaceutical composition. According to anotherembodiment, the pharmaceutical composition comprises a therapeuticamount of a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2(MK2) inhibitor comprising an MK2 polypeptide inhibitor or a functionalequivalent thereof. According to another embodiment, the packagingmaterial is an instruction. According to another embodiment, thecomposition of the kit further comprises a pharmaceutically acceptableexcipient.

According to one embodiment, the described invention provides a methodfor inhibiting kinase activity of a kinase. According to anotherembodiment, the kinase activity is MK2 kinase activity.

According to one embodiment, the described invention provides a methodfor reducing fibroblast proliferation, extracellular matrix deposition,or a combination thereof in a tissue of a subject. According to anotherembodiment, the tissue is cardiac tissue.

According to one embodiment, the described invention provides a methodfor preserving heart function or improving cardiac function aftermyocardial infarction (MI). Cardiac function can be measured bytechniques known to one skilled in the art. Such measurements include,but are not limited to, echocardiography, ejection fraction, fractionalshortening, ventricular volume and end diastolic diameter. Preservingheart function includes, without limitation, maintaining ejectionfraction, maintaining fractional shortening, and decreasing leftventricular dilation compared to an untreated control subject.

According to one embodiment, the described invention provides a methodfor attenuating cardiac dilation after MI.

According to one embodiment, the described invention provides a methodfor protecting cardiomyocytes after MI. Protecting cardiomyocytesincludes, but is not limited to, reducing cardiac fibrosis, musclesparing, and preserving systolic function. Protecting cardiomyocytes canbe determined by techniques available to one of skill in the art. Suchtechniques include, but are not limited to, echocardiography.

According to one embodiment, the described invention provides a methodfor reducing at least one pathology selected from the group consistingof an aberrant deposition of an extracellular matrix protein, anaberrant promotion of fibroblast proliferation in the heart, an aberrantinduction of myofibroblast differentiation, and an aberrant promotion ofattachment of myofibroblasts to an extracellular matrix, compared to anormal healthy control subject.

According to one embodiment, the described invention provides a methodfor reducing fibrosis after MI. According to another embodiment, thereduction in fibrosis is 50%. According to another embodiment, thereduction in fibrosis is greater than 50%. According to anotherembodiment, the reduction in fibrosis is greater than 50%.

According to one embodiment, the described invention provides a methodfor inhibiting apoptosis. According to one embodiment, the methodcomprises inhibiting caspase 3/7 activity. According to anotherembodiment the described invention provides a method for enhancinglactate dehydrogenase (LDH) release. According to another embodiment,the described invention provides a method for inhibiting apoptosis.

According to one embodiment, the described invention provides a methodfor inhibiting heterogeneous nuclear ribonucleoprotein AO (HNRNPA0)expression, activation or a combination thereof.

According to one embodiment, the described invention provides a methodfor enhancing fibroblast cell death. Cell death includes, but is notlimited, necrosis and apoptosis. According to another embodiment, thedescribed invention provides a method for decreasing fibroblastviability. According to another embodiment, the fibroblast is a cardiacfibroblast. According to another embodiment, the cardiac fibroblast is amyofibroblast.

According to one embodiment, the functional equivalent of the MK2polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has a substantial sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1).

According to one embodiment, the functional equivalent of the MK2polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 70 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1). According to another embodiment, the functional equivalent of theMK2 polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 75 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1). According to another embodiment, the functional equivalent of theMK2 polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 80 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1). According to another embodiment, the functional equivalent of theMK2 polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 85 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1). According to another embodiment, the functional equivalent of theMK2 polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 90 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1). According to another embodiment, the functional equivalent of theMK2 polypeptide inhibitor MMI-0100 of amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 95 percent sequenceidentity to the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO:1).

According to one embodiment, the MMI-0100 peptide(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) of the described inventioncomprises a fusion protein in which a cell penetrating peptide (CPP;YARAAARQARA; SEQ ID NO: 2) is operatively linked to a therapeutic domain(TD; KALARQLGVAA; SEQ ID NO: 3) in order to enhance therapeuticefficacy.

Examples of polypeptides functionally equivalent to the therapeuticdomain (TD; KALARQLGVAA; SEQ ID NO: 3) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence KALARQLAVA (SEQ ID NO: 9), apolypeptide of amino acid sequence KALARQLGVA (SEQ ID NO: 10), apolypeptide of amino acid sequence KALARQLGVAA (SEQ ID NO: 11), apolypeptide of amino acid sequence KALNRQLGVAA (SEQ ID NO: 12), apolypeptide of amino acid sequence KAANRQLGVAA (SEQ ID NO: 13), apolypeptide of amino acid sequence KALNAQLGVAA (SEQ ID NO: 14), apolypeptide of amino acid sequence KALNRALGVAA (SEQ ID NO: 15), apolypeptide of amino acid sequence KALNRQAGVAA (SEQ ID NO: 16), apolypeptide of amino acid sequence KALNRQLAVA (SEQ ID NO: 17), apolypeptide of amino acid sequence KALNRQLAVAA (SEQ ID NO: 18), apolypeptide of amino acid sequence KALNRQLGAAA (SEQ ID NO: 19), and apolypeptide of amino acid sequence KALNRQLGVA (SEQ ID NO: 20).

Examples of polypeptides functionally equivalent to the cell penetratingpeptide (CPP; YARAAARQARA; SEQ ID NO: 2) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 21), apolypeptide of amino acid sequence WLRRIKA (SEQ ID NO: 22), apolypeptide of amino acid sequence YGRKKRRQRRR (SEQ ID NO: 23), apolypeptide of amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 24), apolypeptide of amino acid sequence FAKLAARLYR (SEQ ID NO: 25), apolypeptide of amino acid sequence KAFAKLAARLYR (SEQ ID NO: 26), and apolypeptide of amino acid sequence HRRIKAWLKKI (SEQ ID NO: 27).

According to one embodiment, the pharmaceutical composition inhibits akinase activity of a kinase. According to another embodiment, the kinaseactivity is MK2 kinase activity.

According to one embodiment, kinase inhibition may, for example, beeffective to reduce fibroblast proliferation, extracellular matrixdeposition, or a combination thereof in a tissue of a subject. Accordingto another embodiment, the tissue is cardiac tissue.

According to one embodiment, the kinase inhibition may, for example, beeffective to improve cardiac function after myocardial infarction (MI).Cardiac function can be measured by techniques known to one skilled inthe art. Such measurements include, but are not limited to,echocardiography, ejection fraction, fractional shortening, ventricularvolume and end diastolic diameter. Improved cardiac function includes,but is not limited to, increased ejection fraction, increased fractionalshortening, and decreased left ventricular dilation, compared to anuntreated control subject.

According to one embodiment, the kinase inhibition may, for example, beeffective to attenuate cardiac dilation after MI.

According to one embodiment, the kinase inhibition may, for example, beeffective to protect cardiomyocytes after MI. Protection ofcardiomyocytes includes, but is not limited to, a reduction in cardiacfibrosis, muscle sparing, and preservation of systolic function.Protection of cardiomyocytes can be determined by techniques availableto one of skill in the art. Such techniques include, but are not limitedto, echocardiography.

According to one embodiment, the kinase inhibition may, for example, beeffective to reduce at least one pathology selected from the groupconsisting of an aberrant deposition of an extracellular matrix protein,an aberrant promotion of fibroblast proliferation in the heart, anaberrant induction of myofibroblast differentiation, and an aberrantpromotion of attachment of myofibroblasts to an extracellular matrix,compared to a normal healthy control subject.

According to one embodiment, the kinase inhibition may, for example, beeffective to reduce fibrosis. According to another embodiment, thereduction in fibrosis is 50%. According to another embodiment, thereduction in fibrosis is less than 50%. According to another embodiment,the reduction in fibrosis is greater than 50%.

According to one embodiment, the kinase inhibition may, for example, beeffective to inhibit caspase 3/7 activity. According to anotherembodiment, the inhibition may, for example, be effective to enhancelactate dehydrogenase (LDH) release. According to another embodiment,the inhibition may, for example, be effective to inhibit apoptosis.

According to one embodiment, the kinase inhibition may, for example, beeffective to inhibit heterogeneous nuclear ribonucleoprotein AO(HNRNPA0) expression.

According to one embodiment, the kinase inhibition may, for example, beeffective to enhance fibroblast cell death. Cell death includes, but isnot limited, necrosis and apoptosis. According to another embodiment,the inhibition may, for example, be effective to decrease fibroblastviability. According to another embodiment, the fibroblast is a cardiacfibroblast. According to another embodiment, the cardiac fibroblast is amyofibroblast.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to another embodiment, the pharmaceutical composition inhibitsat least 50% of the kinase activity of the kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 65% of thekinase activity of the kinase. According to another embodiment, thepharmaceutical composition inhibits at least 75% of the kinase activityof that kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 80% of the kinase activity of that kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 85% of the kinase activity of that kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 90% of thekinase activity of that kinase. According to another embodiment, thepharmaceutical composition inhibits at least 95% of the kinase activityof that kinase.

According to some embodiments, the pharmaceutical composition inhibits akinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2 kinase). According to some other embodiments, thepharmaceutical composition inhibits at least 50% of the kinase activityof MK2 kinase. According to some other embodiments, the pharmaceuticalcomposition inhibits at least 65% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 75% of the kinase activity of MK2 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 80% of thekinase activity of MK2 kinase. According to another embodiment, thepharmaceutical composition inhibits at least 85% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 90% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 95% of the kinase activity of MK2 kinase.

According to some other embodiments, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.00001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.0001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.001 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.1 mg/kg (or 100 μg/kg) body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 1 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 10 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 2 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 3 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 4 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 5 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 60 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 70 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 80 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 90 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitor peptide of the pharmaceuticalcomposition is of an amount from about 0.000001 mg/kg body weight toabout 90 mg/kg body weight. According to another embodiment, thetherapeutic amount of the therapeutic inhibitor peptide of thepharmaceutical composition is of an amount from about 0.000001 mg/kgbody weight to about 80 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 70 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 60 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 50 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 40 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideis of an amount from about 0.000001 mg/kg body weight to about 30 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 20 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 10 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.01 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.0001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.00001 mg/kg bodyweight.

According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 25 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 1 μg/kg/day to 2 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 2 μg/kg/day to 3 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 3 μg/kg/day to 4 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical ranges from 4μg/kg/day to 5 μg/kg/day. According to some other embodiments, thetherapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 6 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 6 μg/kg/day to 7 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 7 μg/kg/day to 8 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 8 μg/kg/day to 9 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 9 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 5 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 10 μg/kg/day to 15 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 15 μg/kg/day to 20 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 25 μg/kg/day to 30 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 30 μg/kg/day to 35 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 35 μg/kg/day to 40 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 40 μg/kg/day to 45 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 45 μg/kg/day to 50 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 50 μg/kg/day to 55 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 55 μg/kg/day to 60 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 60 μg/kg/day to 65 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 65 μg/kg/day to 70 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 70 μg/kg/day to 75 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 80 μg/kg/day to 85 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 85 μg/kg/day to 90 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 90 μg/kg/day to 95 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 95 μg/kg/day to 100 μg/kg/day.

According to some embodiments, the polypeptide of the inventioncomprises D-amino acids (which are resistant to L-amino acid-specificproteases in vivo), a combination of D- and L-amino acids, and various“designer” amino acids (e.g., β-methyl amino acids, C-α-methyl aminoacids, and N-α-methyl amino acids, etc.) to convey special properties.Examples of synthetic amino acid substitutions include ornithine forlysine, and norleucine for leucine or isoleucine.

According to some embodiments, the polypeptide may be linked to othercompounds to promote an increased half-life in vivo, such aspolyethylene glycol or dextran. Such linkage can be covalent ornon-covalent as is understood by those of skill in the art. According tosome other embodiments, the polypeptide may be encapsulated in a micellesuch as a micelle made ofpoly(ethyleneglycol)-block-poly(polypropylenglycol) orpoly(ethyleneglycol)-block-polylactide. According to some otherembodiments, the polypeptide may be encapsulated in degradable nano- ormicro-particles composed of degradable polyesters including, but notlimited to, polylactic acid, polyglycolide, and polycaprolactone.

According to one embodiment, the carrier of the composition of thedescribed invention includes a release agent, such as a sustainedrelease or delayed release carrier. In such embodiments, the carrier canbe any material capable of sustained or delayed release of thepolypeptide to provide a more efficient administration, e.g., resultingin less frequent and/or decreased dosage of the polypeptide, improvingease of handling, and extending or delaying effects on diseases,disorders, conditions, syndromes, and the like, being treated, preventedor promoted. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, or microcapsules of natural and syntheticpolymers and the like. Liposomes may be formed from a variety ofphospholipids, including, but not limited to, cholesterol, stearylaminesor phosphatidylcholines.

Methods for synthesis and preparation of small peptides are well knownin the art and are disclosed, for example, in U.S. Pat. Nos. 5,352,461;5,503,852; 6,071,497; 6,331,318; 6,428,771 and U.S. Publication No.20060040953. U.S. Pat. Nos. 6,444,226 and 6,652,885 describe preparingand providing microparticles of diketopiperazines in aqueous suspensionto which a solution of active agent is added in order to bind the activeagent to the particle. These patents further describe a method ofremoving a liquid medium by lyophilization to yield microparticlescomprising an active agent. Altering the solvent conditions of suchsuspension to promote binding of the active agent to the particle isdisclosed in U.S. Application Nos. 60/717,524; 11/532,063; and Ser. No.11/532,065; U.S. Pat. No. 6,440,463; and U.S. application Ser. Nos.11/210,709 and 11/208,087. Each of these patents and patent applicationsis incorporated by reference herein.

According to one embodiment, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) and its functional equivalents of the present invention can bedried by a method of spraying drying as disclosed in, for example, U.S.application Ser. No. 11/678,046 (incorporated by reference herein).

According to one embodiment, the polypeptide of the invention may beapplied in a variety of solutions. A suitable formulation is sterile,dissolves sufficient amounts of the polypeptides, and is not harmful forthe proposed application. For example, the compositions of the describedinvention may be formulated as aqueous suspensions wherein the activeingredient(s) is (are) in admixture with excipients suitable for themanufacture of aqueous suspensions.

Such excipients include, without limitation, suspending agents (e.g.,sodium carboxymethylcellulose, methylcellulose,hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth, and gum acacia), dispersing or wetting agents including,a naturally-occurring phosphatide (e.g., lecithin), or condensationproducts of an alkylene oxide with fatty acids (e.g., polyoxyethylenestearate), or condensation products of ethylene oxide with long chainaliphatic alcohols (e.g., heptadecaethyl-eneoxycetanol), or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand a hexitol (e.g., polyoxyethylene sorbitol monooleate), orcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides (e.g., polyethylene sorbitanmonooleate).

Compositions of the described invention also may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil(e.g., arachis oil, olive oil, sesame oil or coconut oil) or in amineral oil (e.g., liquid paraffin). The oily suspensions may contain athickening agent (e.g., beeswax, hard paraffin or cetyl alcohol).

Compositions of the described invention also may be formulated in theform of dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water. The active ingredient insuch powders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients also may bepresent.

According to one embodiment, the described invention providesadministering the pharmaceutical composition to a subject. The step ofadministering comprises administering the composition orally, topically,parenterally, buccally, sublingually, by inhalation, or rectally.

According to one embodiment the administering step comprisesadministering the composition as a single dose post-injury.

According to one embodiment, the administering step comprisesadministering the composition orally. According to another embodiment,the administering step comprises administering the compositiontopically. According to another embodiment, the administering stepcomprises administering the composition parenterally. According toanother embodiment, the administering step comprises administering thecomposition buccally. According to another embodiment, the administeringstep comprises administering the composition sublingually. According toanother embodiment, the administering step comprises administering thecomposition by inhalation. According to another embodiment, theadministering step comprises administering the composition rectally.

According to one embodiment, the composition is in the form of a tablet,a pill, a gel, an injectable solution, an aerosol, a troche, a lozenge,an aqueous suspension, an oily suspension, a dispersible powder, agranule, a bead, an emulsion, an implant, a cream, a patch, a capsule, asyrup, a suppository or an insert. According to one embodiment, thecomposition is in the form of a tablet. According to another embodiment,the composition is in the form of a pill. According to anotherembodiment, the composition is in the form of a gel. According toanother embodiment, the composition is in the form of an injectablesolution. According to another embodiment, the composition is in theform of an aerosol. According to another embodiment, the composition isin the form of a troche. According to another embodiment, thecomposition is in the form of a lozenge. According to anotherembodiment, the composition is in the form of an aqueous suspension.According to another embodiment, the composition is in the form an oilysuspension. According to another embodiment, the composition is in theform of a dispersible powder. According to another embodiment, thecomposition is in the form of a granule. According to anotherembodiment, the composition is in the form of a bead. According toanother embodiment, the composition is in the form of an emulsion.According to another embodiment, the composition is in the form of animplant. According to another embodiment, the composition is in the formof a cream. According to another embodiment, the composition is in theform of a patch. According to another embodiment, the composition is inthe form of a capsule. According to another embodiment, the compositionis in the form of a syrup. According to another embodiment, thecomposition is in the form of a suppository. According to anotherembodiment, the composition is in the form of an insert.

The compositions of the described invention can be administered orally,topically, parenterally, buccally, sublingually, by inhalation orinsufflation (either through the mouth or through the nose), rectally,or by any means known to the skilled artisan. According to someembodiments, the composition of the described invention is a liquidsolution, a suspension, an emulsion, a tablet, a pill, a capsule, asustained release formulation, a delayed release formulation, a powder,or a suppository. The composition can be formulated with traditionalbinders and carriers such as triglycerides.

The composition can be administered in pharmaceutically acceptablesolutions, which may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic agents.

The compositions of the described invention may be in a form suitablefor oral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules or syrups or elixirs. For oral administration in the formof tablets or capsules, the active drug component may be combined withany oral non-toxic pharmaceutically acceptable inert carrier, such aslactose, starch, sucrose, cellulose, magnesium stearate, dicalciumphosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms)and the like.

Moreover, when desired or needed, suitable binders, lubricants,disintegrating agents and coloring agents also may be incorporated inthe mixture. Powders and tablets may be comprised of from about 5 toabout 95 percent of the composition. Suitable binders include starch,gelatin, natural sugars, corn sweeteners, natural and synthetic gumssuch as acacia, sodium alginate, carboxymethylcellulose, polyethyleneglycol and waxes. Among the lubricants there may be mentioned for use inthese dosage forms, boric acid, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrants include starch, methylcellulose,guar gum and the like.

Pharmaceutical compositions intended for oral use can be preparedaccording to any known method, and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents, and preserving agents in order toprovide pharmaceutically elegant and palatable preparations.

Tablets may contain the active ingredient(s) in admixture with non-toxicpharmaceutically-acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques, for example, to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period, to protect thecomposition from oxidation or photodegradation; or for controlledrelease. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed.

Compositions of the described invention also may be formulated for oraluse as hard gelatin capsules, where the active ingredient(s) is(are)mixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or soft gelatin capsules wherein the activeingredient(s) is (are) mixed with water or an oil medium, for example,peanut oil, liquid paraffin, or olive oil.

Liquid form preparations include solutions, suspensions and emulsionswherein the active ingredient(s) is (are) in admixture with excipientssuitable for the manufacture of aqueous suspensions and emulsions. Suchexcipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatidesuch as lecithin, or condensation products of an alkylene oxide withfatty acids, for example, polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample, heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate. Asan example may be mentioned water or water-propylene glycol solutionsfor parenteral injections or addition of one or more coloring agents,one or more flavoring agents, and one or more sweetening agents, such assucrose or saccharin and pacifiers for oral solutions, suspensions andemulsions.

Compositions of the described invention may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil, forexample arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil, such as liquid paraffin. The oily suspensions may contain athickening agent, for example, beeswax, hard paraffin or cetyl alcohol.Sweetening agents, such as those set forth above, and flavoring agentsmay be added to provide a palatable oral preparation. These compositionscan be preserved by the addition of an antioxidant such as ascorbicacid.

Compositions of the described invention may be formulated in the form ofdispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water. The active ingredient in suchpowders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients, for example,sweetening, flavoring and coloring agents also can be present.

The compositions of the invention also may be in the form of anemulsion. An emulsion is a two-phase system prepared by combining twoimmiscible liquid carriers, one of which is disbursed uniformlythroughout the other and consists of globules that have diameters equalto or greater than those of the largest colloidal particles. The globulesize must be such that the system achieves maximum stability. Usually,separation of the two phases will not occur unless a third substance, anemulsifying agent, is incorporated. Thus, a basic emulsion contains atleast three components, the two immiscible liquid carriers and theemulsifying agent, as well as the active ingredient. Most emulsionsincorporate an aqueous phase into a non-aqueous phase (or vice versa).However, it is possible to prepare emulsions that are basicallynon-aqueous, for example, anionic and cationic surfactants of thenon-aqueous immiscible system glycerin and olive oil. Thus, thecompositions of the invention may be in the form of an oil-in-wateremulsion. The oily phase can be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example a liquid paraffin, or amixture thereof. Suitable emulsifying agents may be naturally-occurringgums, for example, gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of the partial esterswith ethylene oxide, for example, polyoxyethylene sorbitan monooleate.The emulsions also may contain sweetening and flavoring agents.

The compositions of the invention also may be formulated as syrups andelixirs. Syrups and elixirs may be formulated with sweetening agents,for example, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations also may contain a demulcent, a preservative, and flavoringand coloring agents. Demulcents are protective agents employed primarilyto alleviate irritation, particularly mucous membranes or abradedtissues. A number of chemical substances possess demulcent properties.These substances include the alginates, mucilages, gums, dextrins,starches, certain sugars, and polymeric polyhydric glycols. Othersinclude acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,propylene glycol, sodium alginate, tragacanth, hydrogels and the like.

For buccal administration, the compositions of the described inventionmay take the form of tablets or lozenges formulated in a conventionalmanner.

There are three general methods of tablet preparation: thewet-granulation method; the dry-granulation method; and directcompression. The method of preparation and the added ingredients areselected to give the tablet formulation the desirable physicalcharacteristics allowing the rapid compression of tablets. Aftercompression, the tablets must have a number of additional attributessuch as appearance, hardness, disintegration ability, appropriatedissolution characteristics, and uniformity, which also are influencedboth by the method of preparation and by the added materials present inthe formulation.

According to one embodiment, the tablet is a compressed tablet (CT).Compressed tablets are solid dosage forms formed with pressure andcontain no special coating. Generally, they are made from powdered,crystalline, or granular materials, alone or in combination withbinders, disintegrants, controlled-release polymers, lubricants,diluents and colorants. According to another embodiment, the tablet is asugar-coated tablet. These are compressed tablets containing a sugarcoating. Such coatings may be colored and are beneficial in covering updrug substances possessing objectionable tastes or odors and inprotecting materials sensitive to oxidation. According to anotherembodiment, the tablet is a film-coated tablet. These Compressed tabletsare covered with a thin layer or film of a water-soluble material.Numerous polymeric substances with film-forming properties may be used.According to another embodiment, the tablet is an enteric-coated tablet.These Compressed tablets are coated with substances that resist solutionin gastric fluid but disintegrate in the intestine. According to anotherembodiment, the tablet is a multiple compressed tablet. These tabletsare made by more than one compression cycle. Layered tablets areprepared by compressing additional tablet granulation on a previouslycompressed granulation. The operation may be repeated to producemultilayered tablets of two or three layers. Press-coated tablets(dry-coated) are prepared by feeding previously compressed tablets intoa special tableting machine and compressing another granulation layeraround the preformed tablets. According to another embodiment, thetablet is a controlled-release tablet. Compressed tablets can beformulated to release the drug slowly over a prolonged period of time.Hence, these dosage forms have been referred to as prolonged-release orsustained-release dosage forms. According to another embodiment, thetablet is a tablet for solution. These Compressed tablets may be used toprepare solutions or to impart given characteristics to solutions.According to some such embodiments, the tablet is an effervescenttablet. In addition to the drug, these tablets contain sodiumbicarbonate and an organic acid such as tartaric acid or citric acid. Inthe presence of water, these additives react, liberating carbon dioxidethat acts as a disintegrator and produce effervescence. According toanother embodiment, the tablet is a buccal and or sublingual tablet.These are small, flat, oval tablets intended for buccal administrationand that by inserting into the buccal pouch may dissolve or erodeslowly. According to another embodiment, the tablet is a molded tabletor tablet triturate.

According to one embodiment, the tablet comprises a compressed corecomprising at least one component of the described formulation and amembrane forming composition. Formulations utilizing membrane formingcompositions are known to those of skill in the art (see, for example,Remington's Pharmaceutical Sciences, 20th Ed., 2000). Such membraneforming compositions may include, for example, a polymer, such as, butnot limited to, cellulose ester, cellulose ether, and celluloseester-ether polymers, an amphiphilic triblock copolymer surfactant, suchas ethylene oxide-propylene oxideethylene oxide, and a solvent, such asacetone, which forms a membrane over the core. The compressed core maycontain a bi-layer core including a drug layer and a push layer.

The term “non-oral administration” represents any method ofadministration in which a composition is not provided in a solid orliquid oral dosage form, wherein such solid or liquid oral dosage formis traditionally intended to substantially release and or deliver thedrug in the gastrointestinal tract beyond the mouth and/or buccalcavity. Such solid dosage forms include conventional tablets, capsules,caplets, etc., which do not substantially release the drug in the mouthor in the oral cavity. It is appreciated that many oral liquid dosageforms such as solutions, suspensions, emulsions, etc., and some oralsolid dosage forms may release some of the drug in the mouth or in theoral cavity during the swallowing of these formulations. However, due totheir very short transit time through the mouth and the oral cavities,the release of drug from these formulations in the mouth or the oralcavity is considered de minimis or insubstantial. Accordingly, it isunderstood that the term “non-oral” includes parenteral, transdermal,inhalation, implant, and vaginal or rectal formulations andadministrations. Further, implant formulations are to be included in theterm “non-oral,” regardless of the physical location of implantation.Particularly, implantation formulations are known which are specificallydesigned for implantation and retention in the gastrointestinal tract.Such implants are also considered to be non-oral delivery formulations,and therefore are encompassed by the term “non-oral.”

The compositions of the described invention may be in the form ofsuppositories for rectal administration of the composition, such as fortreating pediatric fever. The terms “rectal” or “rectally” as usedherein refer to introduction into the body through the rectum whereabsorption occurs through the walls of the rectum. These compositionscan be prepared by mixing the drug with a suitable nonirritatingexcipient such as cocoa butter and polyethylene glycols which are solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum and release the drug. When formulated as asuppository the compositions of the invention may be formulated withtraditional binders and carriers, such as triglycerides.

According to one embodiment, the tablet is a compressed suppository orinsert. For preparing suppositories, a low melting wax such as a mixtureof fatty acid glycerides, such as cocoa butter, is first melted, and theactive ingredient is dispersed homogeneously therein by stirring orsimilar mixing. The molten homogeneous mixture is then poured intoconvenient sized molds, allowed to cool and thereby solidify.

The compositions of the described invention may be in the form of asterile injectable aqueous or oleaginous suspension. Injectablepreparations, such as sterile injectable aqueous or oleaginoussuspensions, may be formulated according to the known art using suitabledispersing or wetting agents and suspending agents.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1, 3-butanediol. A solutiongenerally is considered as a homogeneous mixture of two or moresubstances; it is frequently, though not necessarily, a liquid. In asolution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. A suspension is adispersion (mixture) in which a finely-divided species is combined withanother species, with the former being so finely divided and mixed thatit does not rapidly settle out. In everyday life, the most commonsuspensions are those of solids in liquid water. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. Forparenteral application, particularly suitable vehicles consist ofsolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension also may contain suitablestabilizers or agents, which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The active agent, when it is desirable to deliver it locally, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Pharmaceutical formulations forparenteral administration include aqueous solutions of the activecompounds in water-soluble form.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, microencapsulated, and if appropriate, with one or moreexcipients, encochleated, coated onto microscopic gold particles,contained in liposomes, pellets for implantation into the tissue, ordried onto an object to be rubbed into the tissue. Such pharmaceuticalcompositions also may be in the form of granules, beads, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, drops or preparations with protractedrelease of active compounds, in whose preparation excipients andadditives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, or solubilizers are customarilyused as described above. The pharmaceutical compositions are suitablefor use in a variety of drug delivery systems. For a brief review ofmethods for drug delivery, see Langer 1990 Science 249, 1527-1533, whichis incorporated herein by reference.

Injectable depot forms are made by forming microencapsulated matrices ofa described inhibitor in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of inhibitor topolymer and the nature of the particular polymer employed, the rate ofdrug release may be controlled. Such long acting formulations may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Examples of other biodegradable polymers include poly(orthoesters)and poly(anhydrides). Depot injectable formulations also are prepared byentrapping the inhibitor of the described invention in liposomes ormicroemulsions, which are compatible with body tissues.

The locally injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions may be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions that may contain anti-oxidants,buffers, bacteriostats and solutes, which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions, which may include suspending agents andthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline,water-for-injection, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

The pharmaceutical agent or a pharmaceutically acceptable ester, salt,solvate or prodrug thereof may be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action. Solutions or suspensions used for parenteral,intradermal, subcutaneous, intrathecal, or topical application mayinclude, but are not limited to, for example, the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Administered intravenously, particularcarriers are physiological saline or phosphate buffered saline (PBS).

The compositions of the described invention may be in the form of adispersible dry powder for delivery by inhalation or insufflation(either through the mouth or through the nose). Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures ofwhich are incorporated by reference. The composition of the describedinvention is placed within a suitable dosage receptacle in an amountsufficient to provide a subject with a unit dosage treatment. The dosagereceptacle is one that fits within a suitable inhalation device to allowfor the aerosolization of the dry powder composition by dispersion intoa gas stream to form an aerosol and then capturing the aerosol soproduced in a chamber having a mouthpiece attached for subsequentinhalation by a subject in need of treatment. Such a dosage receptacleincludes any container enclosing the composition known in the art suchas gelatin or plastic capsules with a removable portion that allows astream of gas (e.g., air) to be directed into the container to dispersethe dry powder composition. Such containers are exemplified by thoseshown in U.S. Pat. Nos. 4,227,522; 4,192,309; and 4,105,027. Suitablecontainers also include those used in conjunction with Glaxo's Ventolin®Rotohaler brand powder inhaler, Fison's Spinhaler® brand powder inhaler.Another suitable unit-dose container which provides a superior moisturebarrier is formed from an aluminum foil plastic laminate. Thepharmaceutical-based powder is filled by weight or by volume into thedepression in the formable foil and hermetically sealed with a coveringfoil-plastic laminate. Such a container for use with a powder inhalationdevice is described in U.S. Pat. No. 4,778,054 and is used with Glaxo'sDiskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237).According to one embodiment, a MicroDose Dry Powder Inhaler (DPI)comprising a piezoelectric vibrator to deaggregate the drug powderpackaged in either moisture-resistant aluminum or plastic blisters,which are pierced with small needles prior to dosing to create openingsinto the flow channel of the device is employed. (MicroDose DPI DrugDelivery, www.ondrugdelivery, Ltd., pp. 26-29 (August 2007). Each ofthese references is incorporated herein by reference.

The compositions of the described invention also may be deliverabletransdermally. The transdermal compositions may take the form of creams,lotions, aerosols and/or emulsions and can be included in a transdermalpatch of the matrix or reservoir type as are conventional in the art forthis purpose. The term “topical” refers to administration of aninventive composition at, or immediately beneath, the point ofapplication. The phrase “topically applying” describes application ontoone or more surfaces(s) including epithelial surfaces.

Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis deviceswhich are prepared according to techniques and procedures well known inthe art. The terms “transdermal delivery system”, transdermal patch” or“patch” refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for post-menopausalindications, and nicotine for smoking cessation. Patches suitable foruse in the described invention include, but are not limited to, (1) thematrix patch; (2) the reservoir patch; (3) the multi-laminatedrug-inadhesive patch; and (4) the monolithic drug-in-adhesive patch;TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS, pp. 249-297 (Tapash K.Ghosh et al. eds., 1997), hereby incorporated herein by reference. Thesepatches are well known in the art and generally available commercially.

The compositions of the described invention may further includeconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral application whichdo not deleteriously react with the active compounds. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil; fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, etc.

The compositions may be sterilized and if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. For parenteralapplication, suitable vehicles include solutions, such as oily oraqueous solutions, as well as suspensions, emulsions, or implants.Aqueous suspensions may contain substances which increase the viscosityof the suspension and include, for example, but not limited to, sodiumcarboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension also may contain stabilizers. These compositions also maycontain adjuvants including preservative agents, wetting agents,emulsifying agents, and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It also may be desirable to include isotonic agents, forexample, sugars, sodium chloride and the like. Prolonged absorption ofthe injectable pharmaceutical form may be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

The composition, if desired, also may contain minor amounts of wettingor emulsifying agents or pH buffering agents. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable buffering agents include,without limitation: acetic acid and a salt (1%-2% w/v); citric acid anda salt (1%-3% w/v); boric acid and a salt (0.5%-2.5% w/v); andphosphoric acid and a salt (0.8%-2% w/v). Suitable preservatives includebenzalkonium chloride (0.003%-0.03% w/v); chlorobutanol (0.3%-0.9% w/v);parabens (0.01%-0.25% w/v) and thimerosal (0.004%-0.02% w/v).

The pharmaceutical compositions within the described invention contain atherapeutically effective amount of an MK2 inhibitor compound andoptionally other therapeutic agents included in apharmaceutically-acceptable carrier. The components of thepharmaceutical compositions also are capable of being commingled in amanner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficiency.

The therapeutically effective amount of the MK2 inhibitor compound maybe provided in particles. The particles may contain the therapeuticagent(s) in a core surrounded by a coating. The therapeutic agent(s)also may be dispersed throughout the particles. The therapeutic agent(s)also may be adsorbed into the particles. The particles may be of anyorder release kinetics, including zero order release, first orderrelease, second order release, delayed release, sustained release,immediate release, etc., and any combination thereof. The particle mayinclude, in addition to the therapeutic agent(s), any of those materialsroutinely used in the art of pharmacy and medicine, including, but notlimited to, erodible, nonerodible, biodegradable, or nonbiodegradablematerial or combinations thereof. The particles may be microcapsulesthat contain the therapeutic agent(s) in a solution or in a semi-solidstate. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials may be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired.Bioadhesive polymers of particular interest include bioerodiblehydrogels as described by Sawhney et al in Macromolecules (1993) 26,581-587, the teachings of which are incorporated herein. These includepolyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems.In order to prolong the effect of a drug, it often is desirable to slowthe absorption of the drug from subcutaneous, intrathecal, orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Use of long-term sustained release formulations may be particularlysuitable for treatment of chronic conditions. Long-term sustainedrelease formulations are well-known to those of ordinary skill in theart and include some of the release systems described above.

Depending upon the structure, the MK2 inhibitor compound, and optionallyat least one other therapeutic agent, may be administered per se (neat)or, depending upon the structure of the inhibitor, in the form of apharmaceutically acceptable salt. The MK2 inhibitor compound may formpharmaceutically acceptable salts with organic or inorganic acids, ororganic or inorganic bases. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsconveniently may be used to prepare pharmaceutically acceptable saltsthereof.

By “pharmaceutically acceptable salt” is meant those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell-known in the art. For example, P. H. Stahl, et al. describepharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002).

The salts may be prepared in situ during the final isolation andpurification of the compounds described within the present invention orseparately by reacting a free base function with a suitable organicacid. Representative acid addition salts include, but are not limitedto, acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides, such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides, such asbenzyl and phenethyl bromides, and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsmay be also obtained using standard procedures well known in the art,for example by reacting with a sufficiently basic compound such as anamine with a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which can independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribed the methods and/or materials in connection with which thepublications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the described inventionis not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperatures, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are by weight, molecular weight is weightaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Materials and Methods

MMI-0100 Drug Development

For synthesis of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1),approximately 1 kg of Fmoc-Ala-Wang Resin was transferred into a 50 Lglass solid phase synthesis reaction vessel equipped with a mechanicalstirrer. The resin was allowed to swell in dimethylformide (DMF) for noless than (NLT) 2 hours before draining the DMF. The resin beads thenwere washed with consecutive rinses of DMF. The N-terminal protectinggroup (i.e. Fmoc) was removed (de-blocking step) by treatment with 20%piperidine in DMF and the resin was washed with DMF. The next amino acidin the sequence was coupled in the presence of 1-hydroxybenzotriazole(HOBt) and diisopropylcarbodiimide (DIC). Generally, 2.5-3.5 molarequivalents of Fmoc-amino acid (Fmoc-AA) to the synthesis scale wereused for coupling. The Fmoc-AA was dissolved in DMF and activated by theaddition of HOBt and DIC. The completion of each coupling was monitoredby the Ninhydrin test. If a coupling was incomplete, a second couplingwith the same amino acid was performed by using the symmetricalanhydride method. Generally, 3.0-6.0 molar equivalents of Fmoc-AA to thesynthesis scale were used for coupling. The Fmoc-AA was dissolved indichloromethane (DCM) and a minimal volume of DMF and activated throughthe addition of DIC in a molar ratio of Fmoc-AA/DIC=1.0/0.5. When thefull peptide sequence was completed, the peptide resin was rinsedthoroughly with successive washes of DMF and MeOH. The resin then wasdried under vacuum for NLT 3 hours. Typical recovery of the total driedpeptide resin was approximately 2,800 grams, representing a peptideresin yield of ˜65%.

Approximately 370-500 grams of peptide resin then were transferred intoa suitably sized glass bottle equipped with a magnetic stir bar. Theflask containing the peptide resin was cooled in an ice/water bath or ina refrigerator for no longer than 30 minutes. The trifluoroacetic acid(TFA) cocktail (a mixture of TFA, TIS, and water in the ratio of 95mL:2.5 mL:2.5 mL) was pre-chilled in an ice/water bath for no longerthan 30 minutes. Approximately 8-12 mL of TFA cleavage cocktail per gramof resin was added to this vessel. As soon as the peptide resin and TFAcocktail were combined, the ice/water bath was removed and the reactionmixture was stirred at room temperature for 2-3 hours. The reactionmixture then was filtered through a coarse glass filter and the resinwas washed two times with 0.5-1.0 mL of TFA per gram of resin per wash.The combined filtrate was collected and the resin was discarded. Thefiltrate was then added to ether that was pre-chilled in a refrigeratorfor less than 30 minutes, in a ratio of 1 mL of filtrate per 10 mLether, to precipitate the cleaved peptide. The peptide-ether mixture wasequilibrated to room temperature for no longer than 30 minutes. Theprecipitated peptide was collected on a medium glass filter. Theprecipitate was washed thoroughly with cold ether three times, usingenough ether to at least cover all the precipitate on the filter. Theether then was eluted through the same medium glass filter. The crudepeptide was transferred into a plastic bottle and was placed in adesiccator connected to a mechanical vacuum pump to dry for no laterthan 12 hours. After drying, the crude peptide was stored at 5±3° C. Thecleavage procedure was repeated multiple times until all the peptideresin was cleaved. A typical batch recovery of total dried crude peptidewas approximately 1,250 grams, representing a cleavage yield ofapproximately 110%.

The crude peptide from cleavage was prepared for high-performance liquidchromatography (HPLC) purification by dissolving the peptide in HPLCbuffer at a final crude peptide concentration of 20 mg/mL. The peptidesolution was filtered through a 1 m glass filter membrane and loadedonto a C18 reverse phase column, which was operated by a preparativeHPLC system. The column was washed and equilibrated. A linear gradientwas used to elute the crude peptide from the column. Following eachcrude purification, the fractions were analyzed by an analytical HPLCsystem using a Kromasil C18, 5 rpm, 100 Å 4.6×250 mm column. Fractionsgenerated from the initial purification were pooled based on the HPLCpurity and impurity profile of each fraction. Peptide pools were storedat 2-8° C. until further processing. This process was repeated until allof the crude peptide was purified through the HPLC column and met theMain Pool purity criteria. A salt exchange to acetate salt was performedby HPLC. The final peptide solution was filtered through a 0.22 m filterand loaded onto a tray lyophilizer. The peptide was pre-frozen at 40° C.for no longer than 720 minutes before starting the lyophilization cycle.The lyophilization took approximately 5 days. Approximately 50-55% finalyield resulted from the purification and lyophilization steps.

Cell Permeant Peptide Synthesis and Delivery.

The MMI-0100 peptide (MW=2283.67 g/mol; YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) was synthesized using standard Fmoc chemistry as previouslydescribed [Ward B, Seal B L, Brophy C M, Panitch A. Design of abioactive cell-penetrating peptide: when a transduction domain does morethan transduce. J Peptide Sci. 2009; 15:668-74]. MMI-0100 was preparedand delivered daily intraperitoneally in PBS (50 μg/kg), as previouslydescribed [Vittal R, Fisher A, Gu H, Mickler E A, Panitch A, Lander C,et al. Peptide-mediated Inhibition of MK2 Ameliorates Bleomycin-InducedPulmonary Fibrosis. Am J Respir Cell Mol Biol. 2013]. In cell linestudies, the peptide was dissolved in DMSO before adding to the cellmedia (final [0.5%] to target peptide intracellularly), as previouslydescribed [Ward B C, Kavalukas S, Brugnano J, Barbul A, Panitch A.Peptide inhibitors of MK2 show promise for inhibition of abdominaladhesions. The Journal of surgical research. 2011; 169:e27-36], to givea final MMI-0100 concentration of 20 μM or 100 μM.

Animals and Myocardial Infarction (MI) Model.

Eight to ten week old C57BL/6 mice (25-30 g) were obtained from CharlesRiver (Wilmington, Mass.) and maintained for at least 7 days with freeaccess to standard rodent food and water. Myocardial infarction wasinduced by permanent ligation of the left anterior descending (LAD)coronary artery as described previously [Maejima Y, Kyoi S, Zhai P, LiuT, Li H, Ivessa A, et al. Mstl inhibits autophagy by promoting theinteraction between Beclinl and Bcl-2. Nature Medicine. 2013;19:1478-88; Qian L, Huang Y, Spencer C I, Foley A, Vedantham V, Liu L,et al. In vivo reprogramming of murine cardiac fibroblasts into inducedcardiomyocytes. Nature. 2012; 485:593-8]. Post-surgery, mice wereimmediately treated with lidocaine (6 mg/kg IM) and atropine (0.04-0.10mg/kg IM) upon surgical closure, followed by lidocaine and atropineevery 2-4 hours for the first 24 hours to prevent arrhythmias.Post-anesthesia, mice were given 0.1 mg/kg buprenorphine every 12 hoursfor the first 48 hours. Within the first hour post-MI, 50 μg/kg/dayMMI-0100 peptide (or PBS control) was given intraperitoneally andrepeated for a total of 14 days. In parallel, control groupsunderwent: 1) a sham operation that included every step except thecoronary artery ligation; 2) Daily MMI-0100 intraperitoneally for 14days. Cardiac function was measured by conscious echocardiography usinga Vevo 2100 ultrasound biomicrscopy system (VisualSonics, Inc., Toronto,Canada) at baseline, 1, 7, and 14 days, as previously described [OakleyR H, Ren R, Cruz-Topete D, Bird G S, Myers P H, Boyle M C, et al.Essential role of stress hormone signaling in cardiomyocytes for theprevention of heart disease. Proc Natl Acad Scie USA. 2013;110:17035-40; Willis M S, Homeister J W, Rosson G B, Annayev Y, HolleyD, Holly S P, et al. Functional redundancy of SWI/SNF catalytic subunitsin maintaining vascular endothelial cells in the adult heart.Circulation Res. 2012; 111:el 11-22; Willis M S, Schisler J C, Li L,Rodriguez J E, Hilliard E G, Charles P C, et al. Cardiac muscle ringfinger-1 increases susceptibility to heart failure in vivo. CirculationRes. 2009; 105:80-8].

Histological Analysis of Fibrosis.

Mice were euthanized by isoflurane and cervical dislocation at day 14,fixed in fresh 4% paraformaldehyde for 24 hours, paraffin embedded,processed, and stained with standard hematoxylin and eosin (H&E) andMasson's trichrome (MT). Starting at the ligation with fully facedtissue, 14-15 levels were cut on each block at 50 am (one slide for H&E,one for MT, and 3 unstained; 50 am skipped and then repeated). Controlswere similarly cut starting at a comparable level. The area of fibrosiswas analyzed in 3-4 blindly chosen hearts, each heart at 14-15 levels(point of ligation to apex), 3 sections at each level. Analysis ofcollagen was performed blinded to treatment on these 42-45 sections perheart. Slides were scanned using an Aperio Scanscope (AperioTechnologies, Vista, Calif.) and analyzed using Aperio Imagescope. TheAlgorithm Positive Pixel Count v9 was used to measure the Masson'strichrome staining of collagen (representing both fibrosis and collagenin extracellular matrix), hue value (0.66) and hue width (0.1) were usedanalyzed the tissue outlined using the pen tool. Each section wasanalyzed and exported. The N positive/N total value (representing the %collagen of the entire section) was used to determine a weighted averagefor each slide.

Cell Culture of Primary Cardiac Fibroblast Cells and Cardiomyocyte CellLines.

H9C2 is a myoblast cell line derived from rat myocardium obtained fromATCC® (CRL-1446, ATCC, Manassas, Va.) and cultured according to therecommended protocols. Briefly, cells were maintained at 37° C. with 5%CO₂ in DMEM supplemented with 10% fetal bovine serum and antibiotics(100 U/ml penicillin, 100 mg/ml streptomycin) and split at a ratio of1:4 using 0.05% trypsin every 36 hours. HL-1 cells were obtained fromDr. William Claycomb and cultured according to the published protocols[Claycomb W C, Lanson N A, Jr., Stallworth B S, Egeland D B, Delcarpio JB, Bahinski A, et al. HL-1 cells: a cardiac muscle cell line thatcontracts and retains phenotypic characteristics of the adultcardiomyocyte. Proc. Natl Acad. Sci. USA. 1998; 95:2979-84; White S M,Constantin P E, Claycomb W C. Cardiac physiology at the cellular level:use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cellstructure and function. Am. J. Physiol. Heart Circul. Physiol. 2004;286:H823-9]. Briefly, cells were cultured in Claycomb medium (JRHBiosciences, USA) supplemented with 10% fetal bovine serum (JRHBiosciences), 2 mM L-glutamine (Gibco, Grand Island, N.Y.), 100 LMnorepinephrine (Sigma, USA), 100 U/mL penicillin, and 100 g/mLstreptomycin (Gibco) in flasks precoated with fibronectin and gelatin(Sigma), then incubated at 37° C. in 5% CO2. Cells were split at a ratioof 1:4 using 0.05% trypsin every 48 hours. Primary cardiac fibroblastswere obtained from 2-4-day-old Sprague Dawley rats, according topreviously described protocols (cat. # LK003300, Worthington BiochemicalCorp., Lakewood, N.J.) [Toraason M, Luken M E, Breitenstein M, Krueger JA, Biagini R E. Comparative toxicity of allylamine and acrolein incultured myocytes and fibroblasts from neonatal rat heart. Toxicology.1989; 56:107-17; LaFramboise W A, Scalise D, Stoodley P, Graner S R,Guthrie R D, Magovern J A, et al. Cardiac fibroblasts influencecardiomyocyte phenotype in vitro. Am. J. Physiol. Cell Physiol. 2007;292:C1799-808]. Harvested fibroblasts were seeded in 10 cm FALCONpolystyrene dishes (BD Biosciences), and incubated for 45 min in DMEMwith 10% fetal bovine serum and antibiotics. Cardiomyocytes that did notattach to the noncoated plates were rinsed away and the remainingfibroblasts were given fresh medium, grown to confluence, trypsinized(0.05%) and passaged twice before being used in experiments.

Induction of Hypoxia and Determination of Cell Death In Vitro andEffects of MMI-0100 Given at the Start of Ischemia Time.

Cells were rinsed in PMS and grown in DMEM (cat. #11966-025, Gibco) for2 hours prior to initiating hypoxia (simulated ischemia). Hypoxia wasinduced by placing cells in a hypoxia chamber (HERACELL 150i, ThermoScientific) in a mixture of 5% CO₂/95% N₂ to attain a 1% oxygenconcentration, according to the manufacturer's instruction. Threeexperimental groups were tested for each cell type: 1) Final [0.5% DMSO;Negative Control]; 2) 20 μM MMI-0100 peptide [in a final 0.5% DMSO]; and3) 100 μM LM MMI-0100 peptide [in a final 0.5% DMSO] at 3 time points.The MMI-0100 peptide was added to the cells at the start of the ischemiatime. Cells were cultured in 12 well plates. At the time of performingthe experiments, all cultures were approximately 70-90% confluent. Threedifferent time points were adopted for each of the three cell linesaccording to the severity of cell death under hypoxia determined by LDHrelease: for H9C2 cell line: 8 hr; 16 hr; 24 hr; for HL-1 cell line: 4hr; 8 hr; 12 hr; for cardiac fibroblast cell line: 16 hr; 32 hr; 48 hr.

Cell death was first determined using an LDH release assay (cat.#630117, Clontech), according to the manufacturer's instructions.Briefly, after MMI-0100 peptide treatment and challenge with hypoxia (ornormoxia controls) conditions, 100 μl of culture media was assayed forLDH release using LDH assay kits; in parallel 100 μl of the Catalyst andthe Dye were assayed and read at 490 nm (CLARIOstar, BMG LABTECH GmbH,Ortenberg, Germany). All data was run in triplicate and presented as apercentage of parallel cells treated with a final of 1% Triton-X-100.Caspase 3/7 activity was next determined using a commercial Caspase 3/7activity kit (Cat. # G8091, Promega, Madison, Wis. 53711) in a 384 wellplate (Cat. #781903, Greiner bio-one) according to the manufacturer'sinstructions. Briefly, cells were harvested in 35 μl ice cold PassiveLysis Buffer (cat. # E194A, Promega), rocked for 5 min at RT, thenstored at −80 C. The resulting cell lysates were centrifuged at 10,000×gfor 10 min. The resulting cell lysates (25 al, with 0.6 ug totalprotein) and Caspase-Glo 3/7 Reagent were added to each well in a 1:1ratio and the luminescence was read (CLARIOstar, BMG LABTECH GmbH).

Immunoblot Analysis of MK2 Activity.

The left over cell lysates from the Caspase activity assay were used forthe Western blots. Cell lysate was first fractionated by SDS-4-10%polyacrylamide gel electrophoresis and transferred to PVDF membranes(cat. #162-0177, Bio-Rad, Berkeley, Calif.). After blocking withrecommended blocking reagents for 1 h at the room temperature, themembranes were incubated overnight at 4° C. with primary antibodies inTBS-T, and then incubated with secondary antibodies conjugated with HRPin TBS-T. HNRNPA0, MAPK2, and phospho-MAPK2 proteins were detected usinganti-HNRNPA0 (cat. # HPA036569, 1:1000, Sigma-Aldrich), anti-MAPKAPK2(cat. # SAB4300553, Sigma-Aldrich), and anti-phospho-MAPKAPK2 (cat. #SAB4300241, 1:1000, Sigma-Aldrich). As a loading control, β-actin wasdetected using anti-pactin (cat. # A2228, 1:6000, Sigma-Aldrich). Goatanti-rabbit IgG (whole molecule)-Peroxidase antibody (cat. # A9169,1:1000, Sigma-Aldrich) anti-Mouse IgG (Fab specific)-Peroxidase antibodyproduced in goat (cat. # A9917, 1:6000, Sigma-Aldrich) were used assecondary antibodies. Lumigen ECL Ultra (cat. # TMA-100, Lumigen,Southfield, Mich.) chemiluminescence was detected using the BioSpectrumImaging System (Biospectrum 510, UVP, Upland, Calif.). Quantity One 1-DAnalysis Software (cat. #170-9600, Bio-Rad Laboratories, Inc., Hercules,Calif.) was utilized for densitometry analysis.

Cytokine Analysis of Cell Media for TNFα, IL-1β, and IL-6.

Cytokine analysis to detect TNFα, IL-1β, and IL-6 was performed foreither mouse (HL1) or rat (H9C2 and primary cardiac fibroblasts) usingLuminex multiplex assays (LUM000, LUM401, LUM406, LUM410, LUR000,LUR401, LUR406, LUR410, R&D Systems, Inc., Minneapolis, Minn.) run on aBio-Plex 200 (Bio-Rad, Hercules, Calif.) according to manufacturer'sprotocol. Standard curves were run in parallel with each experiment.

Statistical Analysis.

SigmaPlot (Systat Software, Inc., San Jose, Calif.) was used todetermine significant statistical differences using a Log-rank(Mantel-Cox) test to determine differences in survival or aKruskal-Wallis one-way ANOVA for both in vivo and in vitro studies ateach terminal time point in experiments run in parallel. If significancewas reached (p<0.05), a post-hoc all pairwise Multiple ComparisonProcedures (Tukey Test) was performed between each of the groups todetermine significance. Significance was defined as p<0.05.

Example 1: Treatment of Acute MI with MMI-0100 Peptide Improves CardiacFunction and Heart Failure Measures In Vivo

In this study, acute myocardial infarction (AMI) was induced in eight toten week old C57BL/6 mice by permanent ligation of the left anteriordescending (LAD) coronary artery in order to determine whether treatmentof AMI with MMI-0100 peptide can improve cardiac function and heartfailure measures in vivo.

Echocardiography was used to follow cardiac function and morphometryprior to the surgical induction of an acute myocardial infarction (AMI)induced by thoracotomy and ligation of the left anterior descendingcoronary artery in C57BL/6 mice. Cardiac function was then followed at 7and 14 days post-AMI (FIG. 1A). Mice were separated into three groups:(1) AMI treated with MMI-0100; (2) AMI untreated; and (3) a controlgroup that underwent thoracotomy and sham ligation. Thirty minutes afterthe complete permanent LAD occlusion, the first dose of MMI-0100 wasgiven at the previously established dose (50 mg/kg given daily [Muto A,Panitch A, Kim N, Park K, Komalavilas P, Brophy C M, et al. Inhibitionof Mitogen Activated Protein Kinase Activated Protein Kinase II withMMI-0100 reduces intimal hyperplasia ex vivo and in vivo. VascularPharmacol. 2012; 56:47-55]), illustrated in FIG. 1B. As expected, notall mice survived the AMI procedure, but no differences in survival wereseen after AMI either with or without the MMI-0100 treatment (FIG. 9A).

Cardiac function of mice in the AMI group had an ejection fractiondepressed 25.9% ((87.6-64.9)/87.6*100) and fractional shorteningdepressed 36.6% ((55.7-35.3/55.7*100) after 2 weeks (FIG. 1C, left)compared to sham-operated control mice. Dilation of left ventricle,measured by left ventricular (LV) volume and LV end diastolic diameterwas found at 1 week and plateaued at 2 weeks when compared to shamoperated control mice (decreased 130% and 88.7% respectively) (FIG. 1C,middle & right columns). AMI mice treated with MMI-0100 demonstratedsignificant improvement compared to untreated AMI mice, havingsignificantly greater function at 1 and 2 weeks after AMI, whichtranslated into a decreased LV dilation (LVID; d decreased 29% (vs. 40%without MMI-0100) and LV Volume; d decreased 88% (vs. 130% withoutMMI-0100 (FIG. 1C). Representative M-mode echocardiographic images ofall three groups are shown in FIG. 1D; fully quantified in Table 2.Treatment of mice with MMI-0100 alone did not have any effect on cardiacfunction (Table 3) or survival (FIGS. 9A-B).

Example 2: Treatment of Acute MI with MMI-0100 Treatment DecreasesCardiac Fibrosis In Vivo

Myocardial infarction triggers an inflammatory reaction that results inthe formation of a scar. Healing from myocardial infarction isassociated with alterations in the left ventricle, including dilationand hypertrophy [Bujak M, Frangogiannis N G. The role of TGF-betasignaling in myocardial infarction and cardiac remodeling.Cardiovascular research. 2007; 74:184-95]. In the early stages of anacute MI, TGF-β has been proposed to play a role in deactivatingmacrophages and suppressing endothelial cell cytokine synthesis [BujakM, Frangogiannis N G. The role of TGF-beta signaling in myocardialinfarction and cardiac remodeling. Cardiovascular research. 2007;74:184-95]. In later stages, TGF-β activates fibroblasts to depositextracellular matrix (collagen) which contributes to left ventricularremodeling by promoting fibrosis in the non-infarcted myocardium, inaddition to the myocardium directly affected by ischemia [Bujak M,Frangogiannis N G. The role of TGF-beta signaling in myocardialinfarction and cardiac remodeling. Cardiovascular research. 2007;74:184-95]. Consequences of this cardiac remodeling driven by TGF-β andfibrosis have been associated with myocardial stiffness and systolic anddiastolic cardiac dysfunction, resulting in reduced cardiac output,heart failure, and arrhythmias [van den Borne S W, Diez J, BlankesteijnW M, Verjans J, Hofstra L, Narula J. Myocardial remodeling afterinfarction: the role of myofibroblasts. Nat Rev Cardiol. 2010; 7:30-7].In this study, AMI mice were used to investigate how MMI-0100 affectedmyocardial remodeling after acute MI.

Acute myocardial infarction (AMI) was induced in eight to ten week oldC57BL/6 mice by permanent ligation of the left anterior descending (LAD)coronary artery and treated as described in Example 1. Hearts wereobtained from AMI mice and histologically analyzed for fibrosis inMasson's trichrome stained sections in a systemic manner (FIG. 2A).Based on 4 hearts, analyzed blinded to treatment and objectively usingcomputer algorithms recognizing fibrosis based on hue, AMI mice whichreceived MMI-0100 peptide exhibited 50% less fibrosis than did untreatedmice (FIG. 2B). That is, based on weighted averages of 168-180 crosssectional areas taken from the point of ligation all the way through theapex, MMI-0100 reduced fibrosis to ˜11% (FIG. 2B). Since Masson'strichrome is a stain designed to detect collagen, which is present to asmall extent in normal healthy hearts, analysis was performed on threecontrol groups: (1) thoracotomy and sham ligation; (2) no surgery and nodrug; and (3) a group given MMI-0100 daily that did not undergo surgery.The extensive analysis of these hearts, paralleling the methods used inthe experimental groups, showed that collagen was present in less than1% of the heart area (normal extracellular matrix and basementmembranes) (FIG. 2B). Taken together, these findings demonstrated thatMMI-0100 peptide significantly reduced the fibrosis response by 50%during the remodeling process, even when given 30 minutes after theischemic insult.

Based on the initial histology results in which a decrease in fibrosiswas observed, a more detailed histological analysis was performed onhearts obtained from AMI mice. Representative histological sections frommultiple individual hearts illustrated two general types of fibrosissparing. First, fibrosis that occurs distant to the site of ischemia inAMI, illustrated in FIG. 2C with arrows, is not found to the same extentin the heart sections treated with MMI-0100 (FIG. 2D), although it isstill present (See single arrow). Second, fibrosis at the site ofinfarction after AMI was generally complete (all fibrotic), whereas whenMMI-0100 peptide was given, islands of viable myocytes (See asterisk,FIG. 2D, top panel) could be identified. When investigated at a highermagnification, the islands of myocyte sparing within the ischemic regionscar were found routinely in hearts where MMI-0100 was given (FIG.10D-F). In contrast, complete fibrosis was seen after AMI in all animalsuniformly in multiple representative sections (FIG. 10A-C). The extentof the sparing varied, being more localized to the endocardium at times(FIG. 10D, 10F, arrows), while being more transmural in others (FIG.10E, see asterisk corresponding to asterisk in FIG. 2D, top panel).Without being bound by theory, these findings suggest that thesignificant reduction in fibrosis by MMI-0100 results in critical musclesparing at the site of ischemic insult, while at the same time reducingthe distant non-ischemic site of fibrosis that contributes to thedetrimental effects on cardiac function, which is consistent with thefunctional findings in the same hearts (FIGS. 1A-D).

Example 3: MMI-0100 Peptide Post-Hypoxia Inhibits CardiomyocyteApoptosis In Vitro by Inhibiting MK2 Activity

In this study, the underlying mechanisms by which MMI-0100 peptidespares cardiomyocyte death within the ischemic region and reducesnon-infarcted myocardial fibrosis were investigated by measuringactivation of caspase 3/7 and LDH release; as well as heterogeneousnuclear ribonucleoprotein AO (HNRNPA0), total MK2, andphosphorylated-MK2 (p-MK2) protein expression.

Cell lines derived from ventricular (H9C2) and atrial (HL1)cardiomyocytes were used to determine cell death in the presence ofMMI-0100 peptide at doses previously shown to suppress MK2 activity (20and 100 m) [Muto A, Panitch A, Kim N, Park K, Komalavilas P, Brophy C M,et al. Inhibition of Mitogen Activated Protein Kinase Activated ProteinKinase II with MMI-0100 reduces intimal hyperplasia ex vivo and in vivo.Vascular pharmacology. 2012; 56:47-55; Ward B C, Kavalukas S, BrugnanoJ, Barbul A, Panitch A. Peptide inhibitors of MK2 show promise forinhibition of abdominal adhesions. The Journal of surgical research.2011; 169:e27-36]. Both HL1 and H9C2 cell lines have been established inmodels of acute myocardial infarction by culturing in anoxic (1% oxygen)conditions that induce cell death [Sun J, Sun G, Meng X, Wang H, Wang M,Qin M, et al. Ginsenoside RK3 Prevents Hypoxia-Reoxygenation InducedApoptosis in H9c2 Cardiomyocytes via AKT and MAPK Pathway.Evidence-based complementary and alternative medicine: eCAM. 2013;2013:690190; Zhang C, Lin G, Wan W, Li X, Zeng B, Yang B, et al.Resveratrol, a polyphenol phytoalexin, protects cardiomyocytes againstanoxia/reoxygenation injury via the TLR4/NF-kappaB signaling pathway.International journal of molecular medicine. 2012; 29:557-63; BukowskaA, Hammwohner M, Sixdorf A, Schild L, Wiswedel I, Rohl F W, et al.Dronedarone prevents microcirculatory abnormalities in the leftventricle during atrial tachypacing in pigs. British journal ofpharmacology. 2012; 166:964-80; Liu S X, Zhang Y, Wang Y F, Li X C,Xiang M X, Bian C, et al. Upregulation of heme oxygenase-1 expression byhydroxysafflor yellow A conferring protection fromanoxia/reoxygenation-induced apoptosis in H9C2 cardiomyocytes.International journal of cardiology. 2012; 160:95-101; Severino A,Campioni M, Straino S, Salloum F N, Schmidt N, Herbrand U, et al.Identification of protein disulfide isomerase as a cardiomyocytesurvival factor in ischemic cardiomyopathy. Journal of the AmericanCollege of Cardiology. 2007; 50:1029-37]. Activation of caspase 3/7 andLDH release were measured as markers of cell death in the presence ofMMI-0100.

When the ventricular H9C2 myocyte-derived cells were challenged with 1%hypoxia (FIG. 3A), caspase 3/7 activity increased <5 fold at 8 hourscompared to cells harvested at the start of hypoxia challenge. At 16 and24 hours, caspase 3/7 increased 15-20 fold (FIG. 3B), parallelingincreased LDH release of 60-80% in the same cells (FIG. 3C). The 100 LMMMI-0100 inhibited caspase 3/7 activity at 16 and 24 hours. Withoutbeing bound by theory, this data suggests that MMI-0100 inhibitedapoptotic pathways at this time point (FIG. 3B). Both 20 and 100 μMMMI-0100 significantly enhanced LDH release at 8 hours in cellchallenged with 1% hypoxia, while only the 100 LM MMI-0100 concentrationinduced enhanced LDH release at 16 hours (FIG. 3B). Subsequent studiesto determine the effect of MMI-0100 on LDH release in H9C2 cells innormoxic conditions demonstrated that MMI-0100 did not enhance LDHrelease in the absence of hypoxia at 8 hours (FIG. 11A). This dataconfirmed that MMI-0100 does not enhance LDH release directly, whileparallel studies investigating MMI-0100 effects on the LDH assay itselffound that MMI-0100 had no effect on the colorimetric assay itself (datanot shown).

The effects of MMI-0100 peptide on heterogeneous nuclearribonucleoprotein AO (HNRNPA0), total MK2, and p-MK2 expression in H9C2cells at all time-points tested in the caspase 3/7 activity and LDHrelease studies (FIGS. 4A-B) also were investigated. MMI-0100 at aconcentration of 100 μM significantly inhibited HNRNPA0 expression afterbeing induced by hypoxia (FIG. 4A). Total MK2 and p-MK2 protein levelswere not changed by MMI-0100 peptide at either 20 or 100 LMconcentrations (FIG. 4B).

When the atrial HL1 myocyte-derived cells were challenged with 1%hypoxia (FIG. 5A), caspase 3/7 activity increased 6 fold by 4 hourscompared to cells harvested at the start of hypoxia challenge (FIG. 5B).At 8 and 12 hours, caspase 3/7 increased approximately 10 fold (FIG.5B); paralleling increases in LDH release of 20-60% in the same cells(FIG. 5C). MMI-0100 at a concentration of 100 μM inhibited caspase 3/7activity at 12 hours. Without being bound by theory, this data suggeststhat MMI-0100 inhibited apoptotic pathways at this time point (FIG. 5B).Both 20 and 100 LM MMI-0100 significantly enhanced LDH release at 4hours in cells challenged with 1% hypoxia, while only the 100 LMMMI-0100 had enhanced LDH release at 8 hours (FIG. 5C). Subsequentstudies to determine the effect of MMI-0100 on LDH release of HL1 cellsin normoxic conditions demonstrated that MMI-0100 did not enhance LDHrelease in the absence of hypoxia at 4 hours. MMI-0100 at aconcentration of 100 μM unexpectedly inhibited LDH release in HL1 cells(FIG. 11B).

The effects of MMI-0100 on heterogeneous nuclear ribonucleoprotein AO(HNRNPA0), total MK2, and p-MK2 expression in HL1 cells at alltime-points tested in the caspase 3/7 activity and LDH release studies(FIGS. 5A-C) also were investigated. MMI-0100 at a concentration of 100μM inhibited HNRNPA0 protein expression at 4 and 8 hours after beinginduced by hypoxia (FIG. 6A). Total MK2 and p-MK2 protein levels werenot changed by MMI-0100 peptide at either 20 or 100 μM MMI-0100concentrations (FIG. 6B). This data confirmed that MMI-0100 does notdirectly enhance LDH release.

Example 4: MMI-0100 Treatment Post-Hypoxia Enhances Primary CardiacFibroblast Cell Death In Vitro Despite Inhibiting MK2 Activity

Fibroblasts are integral to the repair of the heart, contributing to theremodeling process after ischemia, fibrosis, and the progression ofheart failure [Porter K E, Turner N A. Cardiac fibroblasts: at the heartof myocardial remodeling. Pharmacol Ther. 2009; 123:255-78]. By physicaland biochemical interaction with cardiomyocytes and the extracellularmatrix, fibroblasts are position to sense and respond to injury.

In this study, primary cardiac fibroblasts were isolated as previouslydescribed in models of acute MI in culture [Rupp H, Maisch B. Control ofapoptosis of cardiovascular fibroblasts: a novel drug target. Herz.1999; 24:225-31; Sangeetha M, Pillai M S, Philip L, Lakatta E G,Shivakumar K. NF-kappa B inhibition compromises cardiac fibroblastviability under hypoxia. Experimental cell research. 2011; 317:899-909;Chu W, Li X, Li C, Wan L, Shi H, Song X, et al. TGFBR3, a potentialnegative regulator of TGF-beta signaling, protects cardiac fibroblastsfrom hypoxia-induced apoptosis. Journal of cellular physiology. 2011;226:2586-94; Leicht M, Briest W, Holzl A, Zimmer H G. Serum depletioninduces cell loss of rat cardiac fibroblasts and increased expression ofextracellular matrix proteins in surviving cells. Cardiovascularresearch. 2001; 52:429-37] and challenged with 1% hypoxia in thepresence or absence of MMI-0100 peptide to determine the peptide'seffects on cardiac fibroblast cell death. These studies were designed toparallel the cardiomyocyte studies (Example 3), with the exception thatlonger time points were used due to the relative resistance (compared tocardiomyocytes) of cardiac fibroblasts to apoptosis and LDH release.

The results of this study are shown in FIGS. 7A-C. In contrast to thecardiomyocyte-derived cell lines tested above, 100 μM MMI-0100 peptidetreatment significantly enhanced caspase 3/7 activity at 16 and 32 hoursof hypoxia (FIG. 7B). LDH release was significantly enhanced in culturedprimary cardiac fibroblasts at 16, 32, and 48 hours of hypoxia (FIG.7C). Subsequent studies to determine the effect of MMI-0100 on LDHrelease of primary cardiac fibroblasts in normoxic conditionsdemonstrated that MMI-0100 did not enhance LDH release in the absence ofhypoxia at 4 hours (FIG. 11B). MMI-0100 peptide did not change MK2activity, as measured by HNRNPA0 protein levels at 16, 32, or 48 hours(FIG. 8A). Similarly, protein levels of MK2 and p-MK2 did notsignificantly differ in the presence of MMI-0100 compared to hypoxiaalone (FIG. 8B). These data indicate that the MMI-0100 peptide enhancescell death of cardiac fibroblasts in the presence of hypoxia. Withoutbeing bound by theory, decreased fibroblast viability may be onemechanism by which a reduction of fibrosis occurs, leading to thedecreased fibrosis seen in vivo (FIGS. 2A-E). This decrease infibroblasts (and presumably fibrosis), in addition to the inhibitedcardiac cell death afforded cardiomyocytes when treated with MMI-0100(FIGS. 3A-C, FIGS. 4A-B, FIGS. 5A-C, FIGS. 6A-B), may offer twomechanisms by which MMI-0100 can improve cardiac function and attenuatecardiac dilation after AMI (FIGS. 1A-D).

Example 5: Cytokine Analysis of Culture Media for TNFα, IL-1β, and IL-6

The inflammatory activation of p38 MAPK in cardiac fibroblastsstimulates the release of IL-1α, TNFα, and MMP-3 [Tondera C, Laube M,Wimmer C, Kniess T, Mosch B, Grossmann K, et al. Visualization ofcyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitorand confocal laser induced cryofluorescence microscopy at 20K inmelanoma cells in vitro. Biochemical and biophysical researchcommunications. 2013; 430:301-6]. These cytokines can stimulatecardiomyocyte contractility depression and cardiomyocyte secretion ofIL-1, IL-6, and TNFα. Since IL-1, IL-6, and TNFα all directly depresscardiac function and mediate heart failure [El-Menyar A A. Cytokines andmyocardial dysfunction: state of the art. J Card Fail. 2008; 14:61-74],these non-cell death induced effects are critical to the dysfunctionfound in acute MI.

In this study, cytokine analysis of TNFα, IL-1β, and IL-6 was performedfor either mouse (HL1) or rat (H9C2 and primary cardiac fibroblast)culture media using Luminex multiplex assays (LUM000, LUM401, LUM406,LUM410, LUR000, LUR401, LUR406, LUR410, R&D Systems, Inc., Minneapolis,Minn.) analyzed on a Bio-Plex 200 (Bio-Rad, Hercules, Calif.) accordingto manufacturer's protocol.

Briefly, HL1, H9C2 or primary cardiac fibroblast cell culture media wasmixed with cytokine capture antibodies coupled to specific bead sets in96-well filter-bottomed microplates and incubated. After cytokines werebound to their corresponding capture antibody/bead, a Phycoerythrin(PE)-conjugated cytokine-specific detection antibody was used as areporter. The amounts of cytokines bound are proportional to the PEsignals generated for each bead set. Fluorescence levels generated bythe beads and the PE-labeled antibody were analyzed on a Bio-Plex 200using dual laser system as the beads pass through a flow cell. Real timequantitative data was generated for each signal. Standard curves wererun in parallel with each experiment.

TNFα, IL-1β, and IL-6 were undetectable in the culture media of all celltypes at all times tested (data not shown). Without being bound bytheory, either these cytokines were released and utilized much earlierthan assayed, or this may be a limitation of using single cellsuspensions that do not replicate the cross talk between cells (e.g.cardiomyocytes and fibroblasts) that occurs in vivo and plays animportant role in cardiac function.

Example 6: Interaction of MMI-0100 with Other Pharmacologically RelevantEnzymes, Receptors and Channels

MMI-0100 was tested for its potential to inhibit 40 non-kinase enzymeassays, including phosphatases and proteases. Enzyme assays wereperformed in the presence and absence of 100 μM MMI-0100. Interaction ata target was considered significant if a greater than 50% inhibition ofactivity was measured in the presence of MMI-0100. No significantinteraction of MMI-0100 was measured at any of the other pharmacologicaltargets in the panel (data not shown).

In order to determine whether MMI-0100 interaction withpharmacologically relevant receptors would result in pharmacologicalactivity, MMI-0100 was tested for its effects in relevant cell-basedassays. Results of the cell-based assays confirmed that MMI-0100 has nosubstantial effect on pharmacologically relevant receptors in cellularfunctional assays (data not shown).

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-24. (canceled)
 25. A method for treating myocardial infarction (MI)in a subject in need thereof, the method comprising administering to thesubject in need thereof a therapeutic amount of a pharmaceuticalcomposition comprising a polypeptide of amino sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent thereofmade from a fusion between a first polypeptide that is a cell permeableprotein (CPP) selected from the group consisting of a polypeptide ofamino acid sequence YARAAARQARA (SEQ ID NO: 2), WERRIKAWERRIKA (SEQ IDNO: 21), WERRIKA (SEQ ID NO: 22), YGRKKRRQRRR (SEQ ID NO: 23),FAKLAARLYR (SEQ ID NO: 25), and KAFAKLAARLYR (SEQ ID NO: 26), and asecond polypeptide that is a therapeutic domain (TD), and apharmaceutically acceptable carrier, wherein the therapeutic amount iseffective to reduce regions of fibrosis at a site of ischemic insult, toreduce non-ischemic site of fibrosis, or a combination thereof comparedto an untreated control subject suffering from MI.
 26. The method ofclaim 25, wherein the MI is acute myocardial infarction (AMI).
 27. Themethod of claim 25, wherein the therapeutic amount is effective toinhibit MK2.
 28. The method of claim 25, wherein the therapeutic amountis effective to increase ejection fraction.
 29. The method of claim 25,wherein the therapeutic amount is effective to increase fractionalshortening.
 30. The method of claim 25, wherein the therapeutic amountis effective to decrease left ventricular dilation.
 31. The method ofclaim 25, wherein the therapeutic amount is effective to inhibitapoptotic cell death of cardiomyocytes, enhance cell death of cardiacfibroblasts, or a combination thereof.
 32. The method of claim 25,wherein the cardiac fibrosis is reduced by 50% compared to the untreatedcontrol subject.
 33. A method for treating ischemia in a subject in needthereof, the method comprising administering to the subject in needthereof a therapeutic amount of a pharmaceutical composition comprisinga polypeptide of amino sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) ora functional equivalent thereof made from a fusion between a firstpolypeptide that is a cell permeable protein (CPP) selected from thegroup consisting of a polypeptide of amino acid sequence YARAAARQARA(SEQ ID NO: 2), WERRIKAWERRIKA (SEQ ID NO: 21), WERRIKA (SEQ ID NO: 22),YGRKKRRQRRR (SEQ ID NO: 23), FAKLAARLYR (SEQ ID NO: 25), andKAFAKLAARLYR (SEQ ID NO: 26), and a second polypeptide that is atherapeutic domain (TD), and a pharmaceutically acceptable carrier,wherein the therapeutic amount is effective to inhibit caspase activity,enhance lactate dehydrogenase (LDH) release, inhibit heterogeneousnuclear ribonucleoprotein AO (HNRNPA0) expression or any combinationthereof in cardiomyocytes.
 34. The method according to claim 33, whereinthe caspase activity is caspase 3/7 activity.
 35. The method accordingto claim 33, wherein the therapeutic amount is effective to enhancecaspase activity, inhibit lactate dehydrogenase (LDH) release, inhibitheterogeneous nuclear ribonucleoprotein AO (HNRNPA0) expression or anycombination thereof in primary cardiac fibroblasts.